Abstract: In order to optimize the enzymatic preparation technology of peptide from the muscle Takifugu flavidus, the response surface methodology was used to study the preparation technology. Taking the degree of hydrolysis as the index, six kinds of proteases, such as acid protease, alkaline protease, trypsin, neutral protease, flavor protease, papain, was screened by the preparation ability, and flavor protease was identified as the target enzyme for the further experiments. The single factor method was used to investigate the effects of solid-liquid ratio, pH value, hydrolysis time, temperature and enzyme concentration on the preparation of polypeptides from the muscle of Takifugu flavidus. The results showed that temperature had the greatest influence on the degree of hydrolysis, enzyme concentration was the second and pH value was third. The optimum preparation conditions were as follows: Enzyme concentration was 4521.96 U/g, the temperature was 55 ℃, the pH value was 7, the solid-liquid ratio was 1:10, and enzymolysis time was 4 h. Under these conditions, the degree of hydrolysis of protein in muscle was 26.26%, while the predicted value was 26.66%, and the relative error was 1.48%, and the predicted result was basically consistent with actual measurement result. It could be seen that the response surface methodology would be reasonable and feasible to optimize the preparation process of polypeptides.