Semiconducting polymer dots (Pdots) are highly fluorescent nanoparticles with exceptional brightness, fast emission rates, excellent photostability, nonblinking, and nontoxicity. They are promising fluorescent probes for biological and medical applications, offering advantages over traditional dyes and quantum dots. Pdots are composed of semiconducting polymers with a particle size of less than 20–30 nm and a high volume fraction (>50%) of hydrophobic conjugated polymer. They exhibit superior optical properties, including high fluorescence brightness, fast emission rates, and photostability, making them ideal for applications such as fluorescence imaging, biosensing, and drug delivery. Pdots are prepared through various methods, including miniemulsion and reprecipitation techniques. The miniemulsion method produces particles ranging from 40 nm to 500 nm, while the reprecipitation method yields particles of 5–30 nm. Pdots have a spherical shape due to the balance between hydrophobic interactions and surface tension. Their optical properties, including absorption and fluorescence, are influenced by the polymer structure and packing density. Pdots exhibit broad absorption bands and tunable emission colors, with fluorescence quantum yields up to 60%, comparable to inorganic quantum dots. Pdots demonstrate exceptional photostability, with a high number of photons emitted before photobleaching. They show minimal photobleaching and no blinking, which is crucial for high-speed imaging applications. Pdots are also chemically stable in biological environments, resisting degradation by ions and reactive oxygen species. Their nontoxicity and biocompatibility make them suitable for in vivo imaging and drug delivery. Surface functionalization and bioconjugation of Pdots are essential for their biological applications. Techniques such as encapsulation in silica, phospholipid, or PLGA polymers, and coating with polyelectrolytes enhance their stability and functionality. Pdots can be modified with various functional groups for targeted delivery and imaging. Their high brightness and stability make them superior to traditional fluorescent dyes and quantum dots, offering new opportunities in nanobiotechnology and nanomedicine.Semiconducting polymer dots (Pdots) are highly fluorescent nanoparticles with exceptional brightness, fast emission rates, excellent photostability, nonblinking, and nontoxicity. They are promising fluorescent probes for biological and medical applications, offering advantages over traditional dyes and quantum dots. Pdots are composed of semiconducting polymers with a particle size of less than 20–30 nm and a high volume fraction (>50%) of hydrophobic conjugated polymer. They exhibit superior optical properties, including high fluorescence brightness, fast emission rates, and photostability, making them ideal for applications such as fluorescence imaging, biosensing, and drug delivery. Pdots are prepared through various methods, including miniemulsion and reprecipitation techniques. The miniemulsion method produces particles ranging from 40 nm to 500 nm, while the reprecipitation method yields particles of 5–30 nm. Pdots have a spherical shape due to the balance between hydrophobic interactions and surface tension. Their optical properties, including absorption and fluorescence, are influenced by the polymer structure and packing density. Pdots exhibit broad absorption bands and tunable emission colors, with fluorescence quantum yields up to 60%, comparable to inorganic quantum dots. Pdots demonstrate exceptional photostability, with a high number of photons emitted before photobleaching. They show minimal photobleaching and no blinking, which is crucial for high-speed imaging applications. Pdots are also chemically stable in biological environments, resisting degradation by ions and reactive oxygen species. Their nontoxicity and biocompatibility make them suitable for in vivo imaging and drug delivery. Surface functionalization and bioconjugation of Pdots are essential for their biological applications. Techniques such as encapsulation in silica, phospholipid, or PLGA polymers, and coating with polyelectrolytes enhance their stability and functionality. Pdots can be modified with various functional groups for targeted delivery and imaging. Their high brightness and stability make them superior to traditional fluorescent dyes and quantum dots, offering new opportunities in nanobiotechnology and nanomedicine.