Abstract: In this study,hempseed protein was firstly hydrolyzed by four enzymes(alcalase,neutral protease,flavourzyme and papain). Then,hemp seed protein isolate hydrolysis was stepwise hydrolyzed using three different proteases(alcalase+neutral,alcalase+flavourzyme and alcalase+papain). Angiotensin-converting enzyme(ACE)inhibitory activity of hemp seed protein hydrolysates and membrane ultrafiltration fractions was analyzed by HPLC. The results showed that the optimal stepwise hydrolyzed conditions of hemp seed protein were alcalase enzyme addition amount was 8000 U/g,the enzymolysis temperature was 50℃,the enzymolysis time was 4 h,the pH was 10 and neutral enzyme addition amount was 8000 U/g,the hydrolysis temperature was 45℃,the enzymolysis time was 4 h,the pH was 7.0. The degree of hydrolysis(DH)and in vitro ACE inhibitory activity were 74.52% and 82.14% respectively. However,there were not significant differences of ACE inhibitory activity between stepwise enzyme hydrolysis and membrane ultrafiltration fractions.It might be to provide theoretical basis for industrial preparation of anti-hypertensive peptides from hemp.