Abstract: The suitable hydrolysis conditions of walnut protein by Trypsin, Neutral protease alone were researched, and two-step-hydrolysis conditions of walnut protein were optimized by response surface methodology ( RSM) .Results showed that the optimum reaction temperature of Trypsin and Neutral protease were 55℃ and 50℃, and pH values were 8.0 and 7.0.Firstly, Trypsin hydrolyzed for 4 hours under the conditions of 55℃, pH 8.0 and enzyme /substrate 2000 U /g, then Neutral protease hydrolyzed for 2.7 hours under the conditions of 50℃, pH 7.0 and enzyme /substrate 3100 U /g. Under this condition, hydrolysis degree of 36.35% and nitrogen recovery ratio of92.53% could be reached. Compared with those of hydrolyzing by Trypsin, Neutral protease alone, hydrolysis degree was increased by 17.79% and 20.11%, and nitrogen recovery ratio were increased by 27.29% and 29.20%.