In this paper，the toxic mechanisms of 2-mercaptobenzimidazole to lysozyme were investigated using spectroscopic and molecular modeling methods under physiological conditions，the binding characterization of 2-mercaptobenzimidazole(MBI) and lysozyme was analyzed，and the changes of conformation and enzyme activity of lysozyme were explored and simulated.The results showed that 2-mercaptobenzimidazole could effectively quench the intrinsic fluorescence of lysozyme via static quenching.The number of binding sites，binding constant and thermodynamic parameters were measured at different temperatures.2-Mercaptobenzimidazole could spontaneously bind with lysozyme through hydrogen bond and van der Waals forces.The results of docking studies showed that 2-mercaptobenzimidazole could bind into the activity site of lysozyme，which further induced the influence on the conformation and enzyme activity of lysozyme.The study provides important references for exploring the toxic mechanism of 2-mercaptobenzimidazole to lysozyme at the molecular level.