DPP4 inhibitors could treat T2DM. Low-cost and accessible egg yolk protein (EYP) has the potential to produce highly bioactive peptides. Therefore, this study was to explore the novel DPP4 inhibitory peptide in EYP. The optimal protease (alcalase and pepsin) was screened using virtual enzymatic digestion. 61 potential peptides were filtered by ultrafiltration, LC-MS/MS, activity prediction and physicochemical property calculations. Then peptides RYHFPEGL, EYF, KFL, YKF and AAQEKIRYW were obtained by machine learning, BIOPEP database and molecular docking. AAQEKIRYW had outstanding hypoglycemia efficacy by in vitro cellular assay and mice plasma assay, with IC50 36.65 μM. Molecular docking and MD revealed that AAQEKIRYW-DPP4 complex was stably bound to S1 and S2' pockets of protein through hydrophilic (hydrogen bonding and electrostatic interactions) and hydrophobic interactions. It will provide a new insight for high-value utilization of EYP and a reference for the efficient screening and mechanism resolution of highly active peptides.