Quantum dots (QDs), semiconductor nanoparticles with unique photo-physical properties, have become a dominant class of imaging probes and universal platforms for engineering multifunctional nanodevices. They possess versatile surface chemistry and superior optical features, finding initial use in various in vitro and in vivo applications. However, careful engineering of QD probes guided by application-specific design criteria is crucial for their successful transition from proof-of-concept studies to clinical applications. This review outlines major design principles and criteria governing the engineering of novel QD probes for nanomedicine and discusses future directions of QD-focused bionanotechnology research. QDs are semiconductor nanoparticles made from group II/VI or III/V elements, with sizes ranging from 2 to 10 nm. Their physical size leads to quantum confinement, resulting in unique optical properties. QDs are used in biomedical applications such as molecular diagnostics, drug delivery, and in vivo imaging. Their advantages include size-tunable and spectrally narrow light emission, efficient light absorption, improved brightness, and large Stokes shift, which expand the capabilities of fluorescence imaging. QDs also provide a platform for engineering multifunctional nanodevices with capabilities of exploiting multiple imaging modalities or merging imaging and therapeutic functionalities. The design of QD probes involves careful engineering of the QD core, surface chemistry, and functionalization with biomolecules. The QD core defines optical properties and serves as a structural scaffold for nanodevices. Surface engineering is crucial for controlling the photophysical properties of the probe and enabling interaction with biological systems. Various approaches have been developed to produce water-soluble QDs, including ligand exchange, polymer encapsulation, and coordination with metal atoms. These approaches aim to achieve biocompatibility, stability, and functionality for biomedical applications. QD probes are used in in vitro applications such as histological evaluation of cells and tissue specimens, single molecule detection, and real-time tracking. They are also used in molecular pathology for sensitive quantitative molecular profiling of cells and tissues, providing unique identification of individual cell lineages and uncovering molecular signatures of pathological processes. QD probes enable the study of dynamic molecular processes in live cells, offering insights into complex biological mechanisms. Their high brightness and photostability make them suitable for sensitive and robust measurement of biomarker expression levels. However, accurate quantitative analysis of multiple biomarkers requires standardization of image acquisition and processing algorithms. Future advancements in QD-based molecular pathology will focus on highly multiplexed quantitative molecular profiling. Engineering of more compact and sensitive QD probes with outstanding stability and non-fouling properties will remain a major focus of research. Decreasing the band gap by tuning the QD chemical composition might enable shifting QD emission into deep blue or far red regions, while keeping the particle size constant. Additionally, significant probe size reduction can be achieved via engineering of compact organic coating layers and ligands. Substitution of thick shells with thinner zwitterionic coatings, development of monQuantum dots (QDs), semiconductor nanoparticles with unique photo-physical properties, have become a dominant class of imaging probes and universal platforms for engineering multifunctional nanodevices. They possess versatile surface chemistry and superior optical features, finding initial use in various in vitro and in vivo applications. However, careful engineering of QD probes guided by application-specific design criteria is crucial for their successful transition from proof-of-concept studies to clinical applications. This review outlines major design principles and criteria governing the engineering of novel QD probes for nanomedicine and discusses future directions of QD-focused bionanotechnology research. QDs are semiconductor nanoparticles made from group II/VI or III/V elements, with sizes ranging from 2 to 10 nm. Their physical size leads to quantum confinement, resulting in unique optical properties. QDs are used in biomedical applications such as molecular diagnostics, drug delivery, and in vivo imaging. Their advantages include size-tunable and spectrally narrow light emission, efficient light absorption, improved brightness, and large Stokes shift, which expand the capabilities of fluorescence imaging. QDs also provide a platform for engineering multifunctional nanodevices with capabilities of exploiting multiple imaging modalities or merging imaging and therapeutic functionalities. The design of QD probes involves careful engineering of the QD core, surface chemistry, and functionalization with biomolecules. The QD core defines optical properties and serves as a structural scaffold for nanodevices. Surface engineering is crucial for controlling the photophysical properties of the probe and enabling interaction with biological systems. Various approaches have been developed to produce water-soluble QDs, including ligand exchange, polymer encapsulation, and coordination with metal atoms. These approaches aim to achieve biocompatibility, stability, and functionality for biomedical applications. QD probes are used in in vitro applications such as histological evaluation of cells and tissue specimens, single molecule detection, and real-time tracking. They are also used in molecular pathology for sensitive quantitative molecular profiling of cells and tissues, providing unique identification of individual cell lineages and uncovering molecular signatures of pathological processes. QD probes enable the study of dynamic molecular processes in live cells, offering insights into complex biological mechanisms. Their high brightness and photostability make them suitable for sensitive and robust measurement of biomarker expression levels. However, accurate quantitative analysis of multiple biomarkers requires standardization of image acquisition and processing algorithms. Future advancements in QD-based molecular pathology will focus on highly multiplexed quantitative molecular profiling. Engineering of more compact and sensitive QD probes with outstanding stability and non-fouling properties will remain a major focus of research. Decreasing the band gap by tuning the QD chemical composition might enable shifting QD emission into deep blue or far red regions, while keeping the particle size constant. Additionally, significant probe size reduction can be achieved via engineering of compact organic coating layers and ligands. Substitution of thick shells with thinner zwitterionic coatings, development of mon