Abstract: This study investigated whether selenium-containing peptides derived from selenium-rich black beans exert inhibitory effects on α-amylase and evaluated their potential hypoglycemic activity in vitro. Selenium-rich black beans from Guangxi, China, were used as raw materials, and selenium-containing peptides were screened and synthesized through a combination of single-factor experiments, response surface methodology, and peptidomics analysis. Four peptides—Se-MetLP, Se-MetIP, TLTSe-MetLR, and Se-CPSFGQ—were identified and evaluated for their α-amylase inhibitory activity. Among the four peptides, Se-MetIP exhibited the highest inhibitory effect, achieving an inhibition rate of 70.58%±2.21% at a concentration of 4 mg/mL, whereas TLTSe-MetLR showed the lowest half-maximal inhibitory concentration (IC₅₀) of 0.47 mg/mL, indicating superior inhibitory potency. Based on their strong α-amylase inhibitory activity, Se-MetIP and TLTSe-MetLR were selected for further investigation of inhibition mechanisms using circular dichroism spectroscopy, isothermal titration calorimetry, and molecular docking analyses, and their in vitro hypoglycemic effects were subsequently evaluated in a hyperglycemic HepG2 cell model. Circular dichroism analysis revealed that peptide binding induced conformational changes in α-amylase, characterized by increased β-sheet content and reduced β-turn and random coil structures, suggesting alterations in the enzyme spatial conformation. Isothermal titration calorimetry demonstrated that peptide-enzyme interactions were exothermic. Molecular docking results showed that the binding free energies of Se-MetIP and TLTSe-MetLR with α-amylase were −5.399 and −6.401 kcal/mol, respectively, with van der Waals forces and hydrogen bonding as the dominant stabilizing interactions, complemented by hydrophobic effects. Furthermore, cellular assays demonstrated that Se-MetIP and TLTSe-MetLR significantly enhanced glucose consumption and promoted glycogen synthesis in insulin-resistant HepG2 cells, thereby improving glucose utilization. Overall, selenium-containing peptides from black beans effectively inhibit α-amylase activity and exhibit in vitro hypoglycemic potential, providing a theoretical basis for the development of hypoglycemic peptides from selenium-rich black beans.