Abstract: In order to achieve the high-value utilization of Miichthys miiuy processing by-products, taking α-glucosidase inhibition rate as an index, we carried out a single factor experiment and applied the response surface methodology to optimize the preparation process of α-glucosidase inhibitory peptides from minced fish muscle of M. miiuy processing by-products. The optimal enzymatic hydrolysis conditions with trypsin were as follows: hydrolysis time 4.8 h, enzyme dosage 0.21%, hydrolysis pH 8.5, material-liquid ratio 1∶2 (m/V), hydrolysis temperature 46 ℃. On this basis, we analyzed the inhibitory activity of the enzymatic hydrolysates before and after the simulated gastrointestinal digestion in vitro, and drew the kinetic curve of enzyme inhibition. Then we separated the enzymatic hydrolysate by SephadexG-25 so as to investigate the molecular weight distribution and amino acid composition of the enzymatic hydrolysates. The results show that the α-glucosidase inhibitory rate increased to 61.79% after that the α-glucosidase inhibitory peptide was simulated gastrointestinal digestion in vitro. Its inhibition type on α-glucosidase was mixed type inhibition. The molecular mass of the enzymatic hydrolysate was concentrated below 3 kD, accounting for 91.85%. After the separation by G-25, the α-glucosidase inhibition rate of F4 fraction was 58.05%. In Group F4, 78.28% of peptides were less than 1 kD. The amino acid analysis shows that the enzymatic hydrolysate was rich in Asp, Glu, Arg, Tyr, Val, Ala, Leu and Lys.