To prepare protein peptides from Hohenbuehelia serotina by enzymatic method, the composition of amino acids, structure and biological activity of protein peptide were analyzed. Proteolytic enzymes were selected with hydrolysis degree and DPPH scavenging rate to optimize the enzymatic hydrolysis conditions by response surface methodology. The protein peptides from Hohenbuehelia serotina was classified to P₁ (＜3.5 kDa), P₂ (3.5～10.0 kDa) and P₃ (＞10.0 kDa), which were analyzed to get the amino acid composition and infrared spectral signature. The antioxidant activity was evaluated by using DPPH, ABTS, OH reduction capacity of free radicals scavenging activity and in vitro simulation of gastrointestinal digestion was also studied. The best hydrolysis conditions as follows: pH 7.6, the reaction time 3.0 h and temperature 42 ℃. The protein peptide P₃ （P＜0.05）had the highest content of total amino acids and the best antioxidant effect. The infrared spectrum peaks of the three components were similar. The activity of the three components did not changed after simulated gastrointestinal digestion. The protein peptides of Hohenbuehelia serotina prepared by enzymatic method are abundant of amino acid, good stability and antioxidant activity. The higher the molecular weight of peptide, the better its antioxidant effect is.