Abstract: In this study,walnut pulp after polyphenol removal was used as raw material,the uniform test was used to optimize hydrolysis conditions of alkali soluble protein extraction,the secondary universal combined rotation design was used to optimize preparation conditions of walnut pulp polypeptides by double enzymes. Simulated gastrointestinal digestive system and simulated gastrointestinal digestive system+mixed lactobacillus system were established by project team at the early stage,which were combined to prepare the proteolytic solution of walnut pulp. The antioxidant activity of alkali solution of walnut pulp prepared by three systems was compared in vitro. The optimal extraction conditions of walnut pulp protein were as follows:pH11,solid-liquid ratio 1:21 (g/mL),water bath extraction at 65 ℃ for 70 min,extraction twice,under this condition the protein content of the extract was 221.6233 mg/g. The process conditions for the preparation of walnut pulp polypeptide by double enzyme method were divided into two stages. The first stage was pepsin enzymolysis,and the optimal conditions were:substrate mass concentration 1 g/100 mL,pH2.4,adding pepsin enzyme amount 133.53 U/g,temperature 40 ℃,and enzymolysis time 139 min. Under this condition,the degree of enzymolysis was 25.90%. Trypsin digestion phase,the second stage,the best conditions were:pH6,adding plus enzyme amount 800 U/g,temperature 20 ℃,and enzymolysis time 240 min. Under this condition,the degree of enzymolysis was 35.57%. Three kinds of total antioxidant capacity of enzymolysis liquid V_C equivalent value were:Enzymolysis solution preparated by double enzymes in vitro 0.0516 mg/mL,enzymolysis solution preparated by gastrointestinal digestion in vitro 0.0634 mg/mL,enzymolysis solution preparated by gastrointestinal digestion in vitro+mixed lactic acid bacteria in gut 0.0411 mg/mL. These three enzymolysis solutiones had antioxidant activity. In the meantime,enzymolysis solution preparated by double enzymes had strong antioxidant activity.