Angiotensin-Ⅰ Converting Enzyme (ACE) is a zinc-containing carboxydipeptidase that regulates blood pressure through renin-angiotensin and kallikrein-kinin systems. The ACE inhibitory peptide (ACEIP) derived from food protein could inhibit the activity of ACE, which is beneficial to antihypertension. In this paper, the inhibition mechanism of the inhibitory peptide LKP from bonito fish on ACE was studied by using fluorescence spectra, ultraviolet absorption spectra, circular dichroism (CD), and isothermal titration calorimetry (ITC) and molecular docking. The fluorescence spectra showed that LKP could effectively quench the endogenous fluorescence of ACE, and the quenching mechanism was static quenching by the formation of a relatively stable complex LKP-ACE. The microenvironment around the tryptophan and tyrosine residues in ACE was localized, decreased the hydrophobicity, and enhanced the polarity. The results of UV and CD showed that the combination of LKP and ACE would lead to the conformation change of ACE. After the addition of LKP, the secondary structure of ACE became looser, and the structural changes of a tightness, loosening and slightly tighter have taken place during the interaction process. The thermodynamic parameters such as enthalpy change (ΔH), entropy change (ΔS), stoichiometric ratio (n) and binding constant (K_a) of the interaction between LKP and ACE were obtained by the ITC method. The results showed that the binding reaction of LKP and ACE was a spontaneous endothermic process driven by entropy, and the binding force was mainly hydrophobic. The stoichiometric ratio (n) value was determined to be about 1, which was enhanced with increasing temperature. At 288, 293 and 299 K, the binding constants Ka of LKP and ACE were 2.2×10³, 0.9×10³ and 5.3×10³, respectively, indicating the affinity of LKP and ACE was relatively low. The results of molecular docking showed that the amino acid residues Gln281 and Lys511 in the S₁ pocket of the ACE active center could form two hydrogen bonds with LKP, and hydrophobic interaction could have occurred between His353 and His513 and LKP, LKP bond to ACE mainly through hydrophobicity, and hydrogen bonds stabilized the spatial structure of the protein. This study provides certain help for exploring the interaction between ACE inhibitory peptide and ACE and offers some theoretical basis for the development of new hypertension drugs.