Biosensing by optical probes is bringing about a revolution in our understanding of physiological and pathological states. Conventional optical probes for biosensing are prone to inaccurate detection results due to various analyte-independent factors that can lead to fluctuations in the absolute signal intensity. Ratiometric optical probes provide built-in self-calibration signal correction for more sensitive and reliable detection. Probes specifically developed for ratiometric optical detection have been shown to significantly improve the sensitivity and accuracy of biosensing. In this review, we focus on the advancements and sensing mechanism of ratiometric optical probes including photoacoustic (PA) probes, fluorescence (FL) probes, bioluminescence (BL) probes, chemiluminescence (CL) probes and afterglow probes. The versatile design strategies of these ratiometric optical probes are discussed along with a broad range of applications for biosensing such as sensing of pH, enzymes, reactive oxygen species (ROS), reactive nitrogen species (RNS), glutathione (GSH), metal ions, gas molecules and hypoxia factors, as well as the fluorescence resonance energy transfer (FRET)-based ratiometric probes for immunoassay biosensing. Finally, challenges and perspectives are discussed.