The best biological enzyme was selected according to the inhibition rate of α-glucosidase. Based on the single factor, optimization of enzymatic hydrolysis conditions could be obtained through the response surface. The enzymatic hydrolysate of the defatted antarctic krill powder was separated and purified by ultrafiltration and G-25. The molecular mass and distribution of enzymatic hydrolysate and peptides were studied by gel chromatography. The results showed that: The compound protease had the best enzymatic hydrolysis effect; The best enzymolysis conditions were enzymolysis time 5.9 h, material-liquid ratio 3.75∶1, enzyme addition amount 0.062 g/g(raw material); The molecular mass of the enzymatic hydrolysate was concentrated below 3 000 u, accounting for 98.63%; The C4 (<3 ku) component by ultrafiltration had the best inhibition rate of α-glucosidase [IC₅₀ was (14.89±2.15) mg/mL]; After purification by G-25, the C4-2 fractions (<3 ku) had higher α-glucosidase inhibitory than other fractions[IC₅₀ was (4.64±0.14) mg/mL]; In the C4-2 fraction, 97.6% of peptides were <1 000 u. The molecular weights of C4-2-1, C4-2-2, and C4-2-3 were 597 u, 335 u, and 246 u respectively. This research for the first time studied the method of preparing α-glucosidase inhibitory active peptide from Antarctic krill powder, which provides a basis for the comprehensive utilization of Antarctic krill resources.