Abstract: : Research on glucose-lowering peptides derived from marine organisms remains relatively limited, and systematic studies on α-glucosidase inhibitory peptides from Trachinotus ovatus are particularly scarce. This study aims to prepare α-glucosidase inhibitory peptide (AGIP) from T. ovatus and investigate their physicochemical properties, thereby laying a theoretical foundation for developing novel hypoglycemic functional foods and promoting the value-added utilization of T. ovatus by-products. We used T. ovatus as the raw material and adopted enzymatic hydrolysis to prepare AGIP. We screened the optimal protease and hydrolysis time based on the α-glucosidase (AG) inhibition rate and degree of hydrolysis (DH) as evaluation indices. Then we investigated the inhibition type, gastrointestinal digestive stability, thermal stability, and acid-base stability of AGIP through enzyme inhibition kinetics, and analyzed its molecular weight distribution and amino acid composition. The results show that the hydrolysate obtained with compound protease after 4 h of hydrolysis exhibited the highest AG inhibition rate of (58.66±0.75)%, with DH of (13.84±0.08)%. The half-maximal inhibitory concentration (IC₅₀) of this hydrolysate toward AG was (20.41±1.75) mg·mL⁻¹, and its inhibition against AG followed a mixed-type inhibition mechanism. After simulated gastrointestinal digestion, the AG inhibition rate of the hydrolysate further increased to 66.55%. The relative molecular mass of 94.14% of the hydrolysate components was below 3kDa, and the content of hydrophobic amino acids (Ala, Pro, Val, Met, Ile, Leu, Phe, and Trp) was relatively high. In conclusion, the α-glucosidase inhibitory peptides prepared in this study demonstrate favorable inhibitory activity and stability, which provides theoretical support for the high-value utilization of T. ovatus and the development of hypoglycemic functional foods.