The objective of this study was to characterize the antioxidant activities， ACE inhibitory activity and functional properties of Lepidotrigla microptera which was hydrolyzed by papain， neutrase， protamex， alcalase and flavourzyme of hydrolysis. High performance liquid chromatograph， ultraviolet-visible (UV) absorption spectroscopy， intrinsic fluorescence spectroscopy and Fourier transform infrared spectroscopy (FTIR) were used to determine the structural changes during hydrolysis. The biological activity of the hydrolysate was characterized by measuring its antioxidant capacity and ACE inhibitory activity. Compared with the other groups， alcalase hydrolysate had the largest proportion of lowest molecular weight (<1 000 u)， and more hydrophobic amino acids， which were 88.2% and 200.5 mg/g， respectively. Structural characterization demonstrated that the surface hydrophobicity， random coil and β-turn of the hydrolysates were significantly higher than those of other groups， which were 69.68%， 23%， respectively. Alcalase hydrolysate showed superior ACE inhibitory activity and antioxidant activity to other hydrolysates， the IC50 were 1.88， 1.92， 1.76， 0.77 mg/mL， respectively. In conclusion， alcalase is the optimal protease for the preparation of Lepidotrigla microptera biological active peptide. This study provided data support and theoretical foundation for diversified development and utilization of Lepidotrigla microptera.