Abstract: In order to improve the resource utilization of Moringa oleifera seeds powder, enzymatic method was used to prepare hypoglycemic peptide from Moringa oleifera seeds. Taking the the degree of proteolysis and α-glucosidase inhibition rate as the response value, single-factor experimental screening and response surface method was used to optimize the optimum enzymatic hydrolysis time, liquid-to-material ratio, hydrolysis pH, enzyme addition, and enzymatic hydrolysis temperature. The hypoglycemic peptide with a molecular weight of <3 kDa were prepared by coarse separation by ultrafiltration, and their inhibitory effects on human liver cancer cells (HepG2 cells) were analyzed by MTT method. The results showed that the optimal conditions of enzymatic hydrolysis were as follows: Time 4.6 h, ratio of liquid to material 40.5:1, pH8.3, enzyme dosage 5.5%, temperature 55 ℃, and the inhibition rate of α-glucosidase was 23.62%±0.14%. The α-glucosidase inhibition rate of the ultrafiltration fraction with a molecular weight of less than 3 kDa had an IC₅₀ value of 5.56 mg/mL. When the concentration of ultrafiltration fraction was 300 μg/mL, the proliferation of HepG2 cells was significantly inhibited after 48 h(P<0.05). The research could lay the foundation for the further separation and purification of hypoglycemic peptide from Moringa oleifera seeds.