Abstract: Walnut peptide was obtained by enzymatic hydrolysis with the walnut powder as raw material.The preparation process of walnut peptide was optimized on foundation of single factor experiment, according to Box-Behnken central composition design principle, the method of response surface analysis was adopted with the peptide mass concentration and degree of hydrolysis as the response value.The effect of p H, temperature, enzyme dosage and time on the peptide mass concentration and degree of hydrolysis were studied in this paper. On the basis of this, different molecular peptides was obtained by enzymatic hydrolysate separated with ultrafiltration system.The antioxidant activity of different molecular peptides was studied.The results showed that the dissolution of protein was sufficient when the walnut-to-water was 1 ∶ 20 g/m L. When the mixed ratio of the alkaline protease and papain was 1∶ 1, the dosage of flavourzyme was 2000 U/g, the hydrolysis effect was better. The optimum preparation conditions were obtained as follows: p H 7.10, temperature 50 ℃, enzyme dosage 8000 U/g and time 3.0 h, the peptide mass concentration was 10.01 mg/m L and the degree of hydrolysis was 11.45%, which was close to the theoretical value.Four kinds of molecular peptides ( ≤5, 5¹0, 10³0 and ≥30 k Da) was obtained by ultrafiltration separation, and both of them had antioxidant activity in vitro.The reducing power ( IC₅₀= 5.47 mg/m L) , the scavenging activity of the DPPH· ( IC₅₀=1.03 mg/m L) , the scavenging activity of the ·OH ( IC₅₀= 5.02 mg/m L) , the scavenging activity of the O-2· ( IC₅₀= 0.68 mg/m L) and the total antioxidant capacity ( 14.18 U/m L) of the ≤5 k Da peptides, was better than the other three kinds of peptides.This study provided certain theoretical guidance for the deep processing and development of walnut products.