Abstract: [Objective] This study aimed to investigate the effects of different reagents for substrate preparation on the detection of plasmin in dairy products,with the goal of establishing an accurate and stable detection method. [Method] A chromogenic substrate method was employed to compare the effects of using ultrapure water versus MTB buffer for plasmin substrate preparation. [Result] The MTB buffer group demonstrated superior performance in terms of absorbance parallelism,spike recovery rate（113%~126%）,and plasmin activity measurements compared to the ultrapure water group （spike recovery rate 141%~152%,exceeding the standard range）.The MTB buffer provided an optimal pH （7.6±0.1）and a stable ionic environment,significantly improving the accuracy and repeatability of the detection. [Conclusion] This study offered an optimized solution for the precise detection of plasmin in dairy products,providing guidance for enhancing quality control in the dairy industry.

Key words: plasmin, microplate reader, MTB buffer, plasmin substrate, dairy product detection