Abstract: The antioxidant peptide was prepared by enzymatic hydrolysis of the protein from the large yellow croaker. Taking reduction force as the response value, the enzymatic dosage, enzymatic hydrolysis temperature, substrate concentration and enzymatic hydrolysis time of neutral protease were optimized by combining single factor and response surface method. The results showed that, among the four enzymes, neutral protease enzymatic hydrolysis had the highest hydrolytic degree (DH) and reducing capacity.The optimal enzymatic hydrolysis process conditions were as follows:The enzyme dosage was 0.4%, the enzymatic hydrolysis temperature was 45℃, the substrate concentration was 25.0%, the enzymatic hydrolysis time was 7 h, and the system pH was 7.0. The reduction force was 0.951.DH of the enzymatic hydrolysate was 37.51%, and the scavenging power (O₂^-·) of superoxide anion radical was 82.42%.SDS-PAGE (polyacrylamide gel electrophoresis) showed that actin disappeared completely after 7 h of enzymatic hydrolysis, and the molecular weights of light chain of hydrolyzed myosin were 27, 15 and 6 kDa.