Abstract: The antioxidant peptides were prepared from Volvariella volvacea protein by protease hydrolysis. Based on the single-factor test, the extraction conditions of antioxidant peptides from V. volvacea protein were optimized using response surface methodology. At the same time, peptides with different retention molecular weights were isolated by ultrafitration, and their antioxidant activity were detected through 2,2-dipheny1-1-picrylhydrazyl (DPPH) radical scavenging activity, Fe²⁺ chelating ability and reducing power. The results showed that the optimum enzyme for enzymatic hydrolysis was neutral protease, and the optimum process for antioxidant peptide was as follows: The enzymatic hydrolysis time was 3.70 h, the enzyme dosage was 3.81%, and the substrate mass concentration was 3.11 g/100 mL. Under the optimal enzymalysis conditions, DPPH free radical scavenging rate of the optimal hydrolysis products was 69.85%±2.52%. The F1 fraction with the lowest molecular weight (<3 kDa) exhibited the highest antioxidant activity. DPPH free radical scavenging rate, Fe²⁺ chelating ability and reducing power of F1 were 78.81%±1.56%, 91.05%±1.65%, 0.47±0.02. The antioxidant peptides from V. volvacea protein can be explored as a potential natural antioxidant.