In this study, a novel method of gradient dilution feeding substrate (GDFS) was established to improve the yield of angiotensin-converting enzyme (ACE) inhibitory peptides from milk protein. The hydrolysis process stability, enzymatic efficiency and kinetics of the method were studied and compared with traditional feeding modes, viz., adding water after feeding substrate or constant concentration feeding substrate. Results showed that the GDFS mode achieved the highest membrane flux and lowest fluctuation of protein concentration in the reactor. Moreover, the GDFS maximized protein conversion rate, yield of peptides, and ACE-inhibitory activity, with their values of 67.58 %, 138.51 g/(g*Neutrase), and 0.74 mg/mL (IC50), respectively. In further study, the kinetic model of GDFS mode was successfully established with K m of 69.481 g/L and V-max of 0.752 g.L-1 min(-1). Based on the optimum condition of the kinetic model, the practical longest runtime was 720 min. Obtained results suggested that GDFS mode could be used as an alternative method in the preparation of high-yield bioactive peptides.