Abstract: The moesin of Larimichthys crocea was in silico digested by gastrointestinal proteases(pepsin and trypsin) of online databases ExPASy PeptideCutter, and the potential bioactive, solubility, absorption, distribution, metabolism, excretion and toxicity of screened tripeptides were predicted and validated in this study. Furthermore, the interaction mechanism of tripeptides and angiotensin-I converting enzyme(ACE) was elucidated by molecular docking and the CDOCKER-Energy was calculated. The ACE inhibitory activity of potential tripeptides in vitro was determined by RP-HPLC and the interaction site of tripeptides in ACE was also clarified. In this paper, four ACE inhibitory tripeptides CMK, GWR, WAK and WQK were successfully identified from the moesin of Larimichthys crocea, with IC₅₀ values of 0.19, 2.40, 0.40, and 1.10 mmol/L respectively. The results of molecular interaction between tripeptides and ACE suggested that tripeptides CMK, GWR, WAK and WQK interacted with key residues Ala354, Glu384 in S₁ pocket of ACE and key residue His353 in S₂ pocket of ACE. Meanwhile, hydrogen bond may attribute to the molecular interaction of tripeptide and the active pocket of ACE. The current study indicated that tripeptides from Larimichthys crocea moesin could be used to develop functional food to prevent hypertension in the future.