In order to prepare dipeptidyl peptidase Ⅳ (DPP-Ⅳ) inhibitory peptide, sturgeon skin collagen was hydrolyzed by protease. A combination of one-factor-at-a-time method and response surface methodology was used optimize the preparation conditions based on percentage of DPP-Ⅳ inhibition. The optimal enzymatic hydrolysis conditions were determined as follows: temperature 50 ℃, solid-to-liquid ratio 1:100 (g/mL), pH 6.12, enzyme dosage 10170.35 U/g, and enzymatic hydrolysis time 12.12 h. The hydrolysate prepared under the optimized conditions was separated and purified by sequential ultrafiltration, gel filtration chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). As identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the amino acid composition of the purified peptide with DPP-Ⅳ inhibitory activity was GPSGLDGAK, and its half-maximum inhibitory concentration (IC₅₀) value was (61.27 ± 1.16) μmol/L. The results of this study can provide a reference for the production of new bioactive components from sturgeon skin.