Abstract: In order to screen peptides with α-glucosidase inhibitory activity from black soybean protein, a dual-enzyme hydrolysis method was employed to prepare α-glucosidase inhibitory peptides from black soybean protein. The peptides were subsequently isolated, identified, and screened using ultrafiltration, liquid chromatography-tandem mass spectrometry (LC-MS/MS), molecular docking and finally verify its in vitro activity. The results demonstrated that after hydrolysis with neutral protease for 180 minutes, followed by hydrolysis with alkaline protease for an additional 180 minutes, the α-glucosidase inhibition rate reached 45.81%±0.51% at a concentration of 2 mg/mL of the black soybean protein hydrolysate. Following ultrafiltration, the fraction I (<1 kDa) exhibited a significantly higher α-glucosidase inhibition rate of 81.29%±1.11% at the same concentration of 2 mg/mL. Within this fraction, a novel α-glucosidase inhibitory peptide, KGLF (Lys-Gly-Leu-Phe) was identified. This peptide showed a binding energy of -3.69 kcal/mol with α-glucosidase in docking studies. The IC₅₀ value of KGLF was determined to be 5.26×10⁻³ mg/mL, which was significantly lower than that of the black soybean protein hydrolysate fraction I. In conclusion, this study successfully prepared and screened highly active hypoglycemic peptides through dual-enzyme hydrolysis of black soybean protein, providing a theoretical basis for the deep processing of black soybean and the development of high-value functional products.