Abstract: This study isolated and characterized bioactive peptides with dipeptidyl peptidase-IV (DPP-IV) inhibitory activity from walnut protein hydrolysates. The hydrolysis process was optimized using response surface methodology with alkaline protease, yielding optimal conditions as follows: Substrate concentration 2.23% (w/v), enzyme dose 9100 U/g, and hydrolysis time 180 min. Under these conditions, the degree of hydrolysis reached 25.47%, and the DPP-IV inhibition rate was 77.85%. The hydrolysate was further separated, purified, and characterized. The fraction with molecular weight <3 kDa (U1) demonstrated the strongest DPP-IV inhibitory activity (IC₅₀=2.81 mg/mL). Subsequent purification of U1 by Sephadex C-25 column chromatography yielded two sub-fractions (F1 and F2), with F2 exhibiting superior activity. A total of 6557 peptides were identified, and five potential hypoglycemic peptides were screened via in silico approaches. Among these, the peptide KFPF showed the highest DPP-IV inhibitory activity (IC₅₀=0.26 mg/mL). Molecular docking simulations revealed that KFPF binds to DPP-IV via six hydrogen bonds and ten hydrophobic interactions. In vitro assays indicated that KFPF acts as a competitive inhibitor. Furthermore, secondary structure analysis by infrared spectroscopy suggested a high content of β-turn (82.03%) and α-helix (15.18%), implying a possible correlation between these structural features and DPP-IV inhibition. These findings demonstrated that KFPF derived from walnut protein has potential as a functional dietary component for blood glucose management.