Abstract: Objective To investigate the mechanism of endogenous fluorescence quenching of pepsin by tinidazole. Methods The fluorescence quenching of pepsin by tinidazole was analyzed by fluorescence spectrometry and UV absorption spectra, under the condition of gastric physiological pH 2.0 (hydrochloric acid-potassium chloride solution). Results The interaction between pepsin and tinidazole was a process of static quenching. The fluorescence quenching constants at different temperatures (288 K, 299 K and 310 K) decreased along with the increase of temperature, while the number of binding sites was about 1. It was found that non-radiation energy transfer occurred during the quenching process, and the type of binding force between pepsin and tinidazole was electrostatic force, with a binding distance of about 5.37 nm. Conclusions These findings provide reference for the study of the mechanism of nitroimidazole in vivo and theoretical basis for further exploration of the mechanism of tinidazole and protein in vivo.