Abstract: To overcome disadvantage of traditional enzymatic hydrolysis and improve reaction effect,ACE- inhibitory peptides from milk protein were prepared by coupling of enzymatic hydrolysis and membrane separation.According to the comparison of protein retention,protease retention,membrane flux,permeates' ACE- inhibitory activity and IC₅₀,the molecular weight of ultra-filtration membrane was determined as 5000 u.The effects of reaction time,substrate concentration,enzymatic dosage and cycle pump speed on conversion ratio of protein were investigated respectively.The optimal conditions obtained from the single factor tests were: substrate concentration7%( W/ W),enzymatic dosage 2000 U / g,reaction time 100 min and cycle pump speed 100 r / min. Under the optimal conditions,conversion ratio of protein reached 53.16% with productivity of 39.59 g peptide·( g protease)^-1 and the inhibitory rate of ACE was 76.81%.Compared with the control group,the three contents were increased by16.99%,16.61% and 18.55% respectively. The relative molecular weight of permeates was measured by high performance size exclusion chromatography( HPSEC). The results showed that the molecular weight could be effectively controlled by ultra- filtration and 94.62% of sample was less than 2253 u. Therefore,it is suggested that the reaction of coupling of enzymatic hydrolysis and membrane separation can provide a more effective method for the preparation of ACE- inhibitory peptides from milk protein.