To promote the release of potential α-glucosidase inhibitory peptides from earthworm protein, earthworm protein hydrolysates were prepared using a combined autolysis–exogenous protease hydrolysis strategy. Potential inhibitory peptides were screened by ultrafiltration, peptidomics, and bioinformatics analysis, followed by in vitro activity evaluation and molecular docking analysis. After 2 h autolysis followed by flavourzyme hydrolysis, the obtained hydrolysate showed an α-glucosidase inhibition rate of 66.59% under optimized conditions of 6000 U·g?1 enzyme dosage, 4 h hydrolysis time, 50 °C, and pH 9.5. The <3 kDa fraction exhibited the strongest inhibitory activity, with an IC50 value of 0.84 mg·mL-1. Six potential α-glucosidase inhibitory peptides were identified by LC-MS/MS combined with bioinformatics analysis. Among them, RGF and FLP showed relatively strong in vitro α-glucosidase inhibitory activities, with IC?50values of 1.56 and 3.57 mM, respectively. Molecular docking analysis revealed that RGF and FLP could interact with key residues of α-glucosidase through hydrogen bonding and hydrophobic interactions, suggesting that their inhibitory activities may be related to binding within the enzyme active pocket. Overall, this study established a preparation process for earthworm-derived α-glucosidase inhibitory peptides and identified bioactive peptide sequences with potential hypoglycemic activity, providing insights into the structure–activity relationships of earthworm-derived peptides and supporting their potential application in functional foods.