There is a close relationship between different biothiols. The changes of one biothiol level in a specific cell metabolism process may be related to the variation of another biothiol, and many diseases are the result of the combination of two or more biothiols. The simultaneous detection of divers biological thiols by fluorescence method is of particular importance for biological research and disease diagnosis. In recent years, breakthrough results have been achieved in this field. The research progress of multi-signal fluorescent probes in the field of simultaneous detection of biothiols is reviewed. According to the objects detected by the probes, the fluorescent probes are systemically classified and discussed, and the corresponding design concept, optical properties, detection mechanism, recognition performance and biological applications are summarized. At the same time, the future development and application in this field are proposed.