Abstract: Alfalfa was used as the material. The effects of neutral protease addition, enzymolysis time, enzymatic hydrolysis temperature and enzymatic hydrolysis pH by single factor experiments were studied and were optimized by the response surface method. The results showed that the optimal enzyme solution were enzyme concentration of 3000 U/g, pH value 8.79, enzymatic hydrolysis at 49.9℃ and enzymolysis time of 4.9 h, and the peptides content was 4.94 mg·mL^-1. Under the optimal enzymolysis conditions, the 1 mg·mL^-1 peptides sample had a strong scavenging capacity against hydroxyl and superoxide anion, the scavenging rate were 74.61% and 72.14%, with weak DPPH radical scavenging capacity. The results indicated that peptides of alfalfa exhibited good antioxidant activity. These results would provide theoretical supports for the further development of alfalfa.