Abstract: In this study, hairtail by-products were used as raw materials to optimize the enzymatic hydrolysis conditions for preparing α-glucosidase (α-GLU) inhibitory peptides using flavourzyme, through single-factor experiments and response surface methodology. The optimal hydrolysis conditions were determined to be an enzymatic hydrolysis temperature of 55 ℃, pH8.0, enzymatic hydrolysis time of 5.3 h, and enzyme addition of 9620 U/g. Under these conditions, the α-GLU inhibition rate of the inhibitory peptide from hairtail waste (IPHW) reached 64.8% ± 0.6%. Molecular weight analysis indicated that IPHW was mainly composed of oligopeptides with molecular weights below 1000 Da, accounting for 89.5%. In vitro stability tests showed that IPHW exhibited high stability under neutral or mildly alkaline pH, non-high-temperature, UV-shielded, and metal ion-free conditions (Na⁺, Mg²⁺, and Al³⁺). After simulated gastrointestinal digestion, IPHW retained more than 85% of its inhibitory activity, demonstrating good digestive stability. These results provide a novel approach for the high-value utilization of hairtail by-products and offer a theoretical basis for the development of natural, safe, and effective α-GLU inhibitory hypoglycemic peptides.