Abstract: The purpose of this study was to prepare a novel Angiotensin-I-Converting Enzyme(ACE) inhibitory peptide by hydrolyzing sheep casein with protease and analyze its molecular inhibitory mechanism, so as to provide technical support for the development of functional sheep milk polypeptide dairy products. In this experiment, sheep casein was used as raw material, with hydrolysis degree, ACE inhibition rate and molecular weight distribution as indexes, the most suitable protease was selected from four proteases, and the components with less than <3 kDa was identified by LTQ Orbitrap Velos mass spectrometer. the potential ACE inhibitory peptides were selected for artificial synthesis and it half inhibitory concentration (IC₅₀) was measured. The inhibition kinetics of enzyme was determined by Linewaver-Burk mapping, and the inhibition mechanism of peptide segment was further explained by molecular docking. The results showed that alkaline protease was the best hydrolytic protease. A new ACE inhibitory peptide KYIPIQY was screened from κ -casein and its IC₅₀ was 5.73 μmol/L. Linewaver-Burk diagram showed that the peptide showed a mixed inhibition mode on ACE. Molecular docking showed that KYIPIQY can form hydrogen bonds with S1 and S2 active pockets of ACE, and closely combine with hydrophobic force. KYIPIQY can distort the Zn²⁺ tetrahedron of ACE, and inhibit the deactivation of ACE catalytic activity, thus exerting high-efficiency ACE inhibitory activity.