Abstract: To prepare the enzymatic hydrolysate of Boletus edulis with high antioxidant activity, the degree of hydrolysis and DPPH· scavenging rate were used as indexes. Protease was used to hydrolyze Boletus edulis. The preparation process of enzymatic hydrolysate was optimized by response surface analysis based on single factor experiment. The results showed that neutral protease was the best enzyme. The optimal process parameters were substrate concentration of 4%, hydrolysis time of 120 min, enzyme dosage of 2500 U/g, and temperature of 40 ℃. Under these conditions, the degree of hydrolysis of the hydrolysate was 34.12%, and the DPPH· scavenging rate was 55.91%. The enzymatic hydrolysate prepared under these conditions had scavenging effects on DPPH·, OH·, and \rmO_2^- \cdot , with IC₅₀ of 0.88, 0.24, and 12.50 mg/mL. The total antioxidant capacity of the optimized hydrolysate was 18.82 U/mL. Under the conditions of enzymatic hydrolysis determined in this study, the hydrolysate of Boletus edulis had antioxidant activity in vitro, which would provide a theoretical basis for the further development of edible fungi protein and polypeptide.