The aim of the experiment was to calculate and analyze the physicochemical properties of three bitter peptides RK7， VN10 and SN12 identified in yak milk hard cheese using bioinformatics methods， and minimize the energy of the peptide structure. Obtained the X-ray diffraction three-dimensional structure of ACE protein crystals from the PDB database， and evaluated and characterized the ACE inhibitory activity of bitter peptides and the mechanism of action with ACE through molecular docking and database comparison. The instability indexes of RK7， VN10 and SN12 were 19.84， 49.44 and 42.87， respectively， and the hydrophobicities were 42.86%， 70.00% and 66.67%， respectively. These three peptides were molecularly docked with ACE， the results of the study showed that the Ala356， His387， Phe391， Pro407， His410， Glu411， Ser517， Val518， and Pro519 residues in the ACE molecule were important active sites for binding to RK7 and VN10， RK7， VN10 and SN12， respectively formed 3， 2 and 0 hydrogen bonds with the amino acids in the active site of ACE. The binding of RK7， VN10 and SN12 to ACE showed similar molecular mechanisms， all of which were bound in the active cavity of ACE(X: 35.46， Y: 43.15， Z: 55.47， R: 9 ?魡). The highest similarities between RK7， VN10 and SN12 and the known ACE inhibitor peptides in the BIOPEP database were 57.14%， 90.00% and 75.00%， respectively. Integrating the interaction of RK7， VN10 and SN12 with the amino acid residues of the active site of ACE and the BIOPEP database comparison results predicted the ACE inhibitory activity of the three peptides: VN10 > RK7 > SN12. Therefore， this experiment provided the reference for studying the ACE inhibitory activity of cheese bitter peptides at the theoretical and molecular levels.