Abstract: In this paper the process conditions of enzymatic hydrolysis to produce the antioxidant peptides from Pholiota nameko protein were studied.Degree of hydrolysis and the hydroxyl radical scavenging rate of hydrolysates were determined to evaluate the antioxidant capacity.According to the hydroxyl radical scavenging activity of hydrolysates, the optimal protease was screened from five kinds of proteases including alcalase, neutral protease, flavor protease, papain and trypsin. The effects of hydrolysis time, substrate concentration and enzyme concentration on the antioxidant activity of the hydrolysates were analyzed with single factor test and the orthogonal test L₉ ( 4³) was used to optimize the factors affecting hydrolysis. The results showed that the activity of alcalase was higher than that of other four kinds of proteases, which could make protein fully hydrolyzed, and the importance of the three factors affecting the hydroxyl radical scavenging activity of antioxidant peptides was in the order of substrate concentration, enzyme concentration and hydrolysis time.The optimized process parameters of alcalase hydrolysis were as follows: substrate concentration 3.0%, enzyme concentration 3500 U/g, p H9.0, temperature 55 ℃, hydrolysis time 3.5 h.Under this condition, the highest hydroxyl radical scavenging rate of the hydrolysate was 82.7% while the degree of hydrolysis was 23.1%.The process technology for preparation of antioxidant peptides from Pholiota nameko protein by alcalase hydrolysis was feasible, and provided technical reference for the development of high value-added products.