The kinetics of casein tryptic hydrolysis to prepare active peptides was investigated. Taking into account the reaction mechanism including single substrate hydrolysis, irreversible enzyme inactivation, and substrate inhibition, a set of exponential equations was established to characterize the enzymatic hydrolysis curves. The verification was carried out by a series of experimental results and indicated that the average regressive error was less than 5%. According to the proposed kinetic model, the kinetic constants and thermodynamic constants of the reaction system were also calculated.