This review summarizes recent advances in the design and application of ratiometric fluorescent probes for the detection of cations, anions, and biomolecules. Ratiometric fluorescence sensing offers high sensitivity and inherent reliability due to the self-calibration provided by monitoring two or more emissions. Small fluorescent molecules have been widely used to develop such probes for sensing, imaging, and biomedical applications. The review highlights several key strategies for ratiometric fluorescent sensing, including internal charge transfer (ICT), excited-state intramolecular proton transfer (ESIPT), fluorescence resonance energy transfer (FRET), through-bond energy transfer (TBET), and monomer-excimer formation. These strategies enable the development of probes that can detect a variety of analytes, including cations, anions, and biomolecules, in both solution and biological samples. ICT-based probes are particularly effective for cation sensing, with changes in absorption and emission spectra indicating the presence of specific ions. ESIPT-based probes are useful for detecting anions and other molecules, with changes in emission bands reflecting analyte interactions. FRET and TBET-based probes are used for detecting metal ions and other analytes, with energy transfer between fluorophores providing a ratiometric signal. Monomer-excimer formation is also used for sensing, with changes in emission characteristics indicating analyte presence. The review also discusses the application of these probes in biological systems, including the detection of thiols, metal ions, and nucleic acids. The probes are designed to be cell membrane permeable and can be used for real-time imaging of intracellular components. Additionally, the review highlights the potential of these probes for use in drug delivery systems and for the detection of biomolecules in living cells. Overall, ratiometric fluorescent probes offer a powerful tool for the quantitative and sensitive detection of various analytes in biological and environmental applications. The review provides an overview of the design principles and applications of these probes, emphasizing their potential for future research and development.This review summarizes recent advances in the design and application of ratiometric fluorescent probes for the detection of cations, anions, and biomolecules. Ratiometric fluorescence sensing offers high sensitivity and inherent reliability due to the self-calibration provided by monitoring two or more emissions. Small fluorescent molecules have been widely used to develop such probes for sensing, imaging, and biomedical applications. The review highlights several key strategies for ratiometric fluorescent sensing, including internal charge transfer (ICT), excited-state intramolecular proton transfer (ESIPT), fluorescence resonance energy transfer (FRET), through-bond energy transfer (TBET), and monomer-excimer formation. These strategies enable the development of probes that can detect a variety of analytes, including cations, anions, and biomolecules, in both solution and biological samples. ICT-based probes are particularly effective for cation sensing, with changes in absorption and emission spectra indicating the presence of specific ions. ESIPT-based probes are useful for detecting anions and other molecules, with changes in emission bands reflecting analyte interactions. FRET and TBET-based probes are used for detecting metal ions and other analytes, with energy transfer between fluorophores providing a ratiometric signal. Monomer-excimer formation is also used for sensing, with changes in emission characteristics indicating analyte presence. The review also discusses the application of these probes in biological systems, including the detection of thiols, metal ions, and nucleic acids. The probes are designed to be cell membrane permeable and can be used for real-time imaging of intracellular components. Additionally, the review highlights the potential of these probes for use in drug delivery systems and for the detection of biomolecules in living cells. Overall, ratiometric fluorescent probes offer a powerful tool for the quantitative and sensitive detection of various analytes in biological and environmental applications. The review provides an overview of the design principles and applications of these probes, emphasizing their potential for future research and development.