Abstract: Taking ovate pompano (Trachinotus ovatus) meat as raw material and taking enzymatic hydrolysate and bile salts in vitro binding rate as index, we screened the optimal protease from five kinds of food protease. Then we used orthogonal design to optimize hydrolysis conditions in order to improve the binding rate of bile acid salts and used GPC method to analyze the molecular weight distribution of peptides. The results show that the tryptic hydrolysates had higher binding rate than that of the other protease hydrolysates, and the in vitro binding rate of sodium cholate, sodium glycocholate and sodium taurocholate by enzymatic protein (100 mg·mL^-1) was 42.1%, 33.5% and 30.1%, respectively, which was 78.3%, 74.4% and 76.8% equivalent to the positive control cholestyramine (20 mg·mL^-1). The maximum percentage of sodium glycocholate hydrate binding rate was obtained under the conditions:hydrolytic time of 2 h, enzyme additives of 2 000 U·g^-1, meal-to-water ratio of 1:4 (g·mL^-1, at pH 8, 37 ℃). Under the optimal condition, the in vitro binding rate of sodium glycocholate hydrate by enzymatic hydrolysate (20 mg·mL^-1) was corresponding to 48.3% of positive control cholestyramine at the same concentration. The relative molecular weight of peptides below 3 kD in enzymolysis products accounted for 77.30%.