Abstract: Polysaccharide was extracted from sea cucmber (Stichopus japonicus) by enzymolysis method, and the enzymatic conditions of papain were optimized through the single factor and response surface methodology by using the yield of polysaccharide as the index. The results showed that the optimal extraction conditions of polysaccharide were as follows:Enzyme dosage 1.60%, pH7.30, temperature 54.0℃. Under these conditions, the yield of polysaccharide was 5.34%. D-glucono-δ-lactone (GDL) was further used for the deproteinization of polysaccharide, and the deproteinization process was optimized with single factor and orthogonal experiment. The optimal conditions for deproteinization were as follows:Reaction temperature 60℃, reaction time 2 h, addition of GDL solution (w/w, 20%) 2.5 mL. The protein removal rate reached 95.8% under optimal conditions. The IC₅₀ of DPPH, hydroxyl free radical, superoxide anion and ABTS⁺ free radical were 0.3, 4.5, 3.9 and 0.85 mg/mL, respectively. When the concentration of sea cucumber polysaccharide was 3.0 mg/mL, ABTS free radical scavenging ability was the strongest, reached 90.4%, and the scavenging ability was similar to that of V_C. Therefore, the polysaccharide would have good antioxidant capacities in vitro.