Abstract: In this study, the mechanism of nitroimidazole tinidazole quenching the endogenous fluorescence of pepsin was studied by fluorescence and UV absorption under the condition of gastric physiological pH 2.0 (hydrochloric acid-potassium chloride solution). It was found that the quenching phenomenon was very obvious, the interaction was a spontaneous exothermic process, and the process was static quenching. The fluorescence quenching constants and the binding sites at different temperatures (288 K, 299 K and 310 K) were calculated. The fluorescence quenching constant decreased with the increase of temperature, and 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 and the binding distance was about 5.37 nm. As a result of the interaction, the microenvironment and conformation of pepsin were changed.