Objective To optimize the preparation process of rice bean polypeptide by the response surface method and analyze its in vitro hypoglycemic activity.Methods The optimal enzyme is screened from the three proteases, and the best enzymatic hydrolysis process conditions are optimized by single factor and response surface experiments. Then, the enzymatically hydrolyzed polypeptide solution is subject to ultrafiltration separation, and the hypoglycemic activities of different components are compared.Results The optimal preparation process conditions for rice bean polypeptide are: basic protease addition amount of 3 860 U/g, substrate concentration of 2.0%, pH of 8.0, enzymatic hydrolysis time of 3 hours, and enzymatic hydrolysis temperature of 60 ℃, resulting in the polypeptide content of 135 mg/g. After ultrafiltration separation of the enzymatically hydrolyzed polypeptide solution, the in vitro hypoglycemic activity is the highest in the F1 (relative molecular weight <3 000) component, with the α-glucosylase inhibition rate of 79.1% and the α-amylase inhibition rate of 81.0% at a mass concentration of 10 mg/mL.Conclusion The rice bean polypeptide prepared by enzymatic hydrolysis with alkaline protease shows a significant in vitro hypoglycemic effect.