This article provides an overview of small molecule-based ratiometric fluorescence probes for detecting cations, anions, and biomolecules. Ratiometric fluorescence sensing is highlighted for its high sensitivity and reliability, achieved through the self-calibration provided by monitoring two or more emissions. The authors discuss various design principles and strategies, including internal charge transfer (ICT), excited-state intramolecular proton transfer (ESIPT), fluorescence resonance energy transfer (FRET), through-bond energy transfer (TBET), and monomer-eximer formation. Each strategy is illustrated with specific examples of probes and their applications in sensing and imaging. The article emphasizes the importance of these probes in addressing a wide range of analytical and imaging challenges, from life sciences to environmental monitoring. The authors also provide a summary of recent advances and future research directions, aiming to inspire further development of new sensing approaches.This article provides an overview of small molecule-based ratiometric fluorescence probes for detecting cations, anions, and biomolecules. Ratiometric fluorescence sensing is highlighted for its high sensitivity and reliability, achieved through the self-calibration provided by monitoring two or more emissions. The authors discuss various design principles and strategies, including internal charge transfer (ICT), excited-state intramolecular proton transfer (ESIPT), fluorescence resonance energy transfer (FRET), through-bond energy transfer (TBET), and monomer-eximer formation. Each strategy is illustrated with specific examples of probes and their applications in sensing and imaging. The article emphasizes the importance of these probes in addressing a wide range of analytical and imaging challenges, from life sciences to environmental monitoring. The authors also provide a summary of recent advances and future research directions, aiming to inspire further development of new sensing approaches.