Biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play important roles in physiological processes, and the detection of thiol using fluorescent probes has attracted attention due to their high sensitivity and selectively and invasive on-time imaging. However, the similar structures and reactivity of these biothiols present great challenges for selective detection. This review focused on the the “aromatic nucleophilic substitution-rearrangement (SNAr-rearrangement) mechanism”, which provided a powerful tool to design fluorescent probes for the discrimination between biothiols. We classify the fluorescent probes according to types of fluorophores, such as difluoroboron dipyrromethene (BODIPY), nitrobenzoxadiazole (NBD), cyanine, pyronin, naphthalimide, coumarin, and so on. We hope this review will inspire exploration of new fluorescent probes for biothiols and other relevant analytes.