Abstract: To efficiently utilize peanut protein resources and explore the hypoglycemic potential of its enzymatic hydrolysates, peanut protein was extracted by alkali dissolution and acid precipitation. Using the α-glucosidase inhibitory rate as the evaluation index, dual-enzyme hydrolysis conditions were optimized via response surface methodology. The results indicated that enzymatic hydrolysates derived from Yuhua 37 (a high-oleic peanut cultivar) possessed superior α-glucosidase inhibitory activity. Sequential hydrolysis of peanut protein was performed using alcalase followed by trypsin, and the optimal conditions were determined as follows: peanut protein mass concentration of 17 mg/mL, total enzyme dosage of 4.7% (mass ratio of the two enzymes was 1∶1), and total hydrolysis time of 156 min (enzyme action time ratio was 5∶1). Under these conditions, the α-glucosidase inhibitory rate of prepared peanut peptides reached 68.41%±0.45% at a peptide concentration of 2 mg/mL, with a corresponding degree of hydrolysis of 25.14%±0.04%. Compared with single-enzyme hydrolysis in preliminary experiments, the dual-enzyme strategy significantly reduced enzyme consumption while maintaining equivalent inhibitory activity. In vitro simulated gastrointestinal digestion exerted variable effects on the α-glucosidase inhibitory activity of peptides from different protein sources; specifically, the inhibitory activity of the self-prepared peanut peptides decreased signifficantly after digestion. In conclusion, the optimized dual-enzymatic hydrolysis process enables efficient preparation of highly active α-glucosidase inhibitory peanuts peptides with lower enzyme usage. However, their inhibitory activity is markedly attenuated after in vitro simulated gastrointestinal digestion.