Bioactive peptides, especially ACE-inhibitory peptides (ACEIPs), have been shown to possess promising application as nutraceuticals. In this study, soluble and insoluble fish protein substrates from leatherjacket (Meuchenia sp.) were hydrolyzed with papain, bromelain and Flavourzyme, and the hydrolysates were used in a systematic screening for ACEIPs. Fractionation of selected <5 kDa hydrolysates obtained from insoluble papain at 6 h (LPI6h), insoluble bromelain at 2 h (LBI2h), insoluble bromelain at 8 h (LBI8h) and insoluble Flavourzyme at 2 h (LFI2h) gave five active fractions (LPI5, LPI6, LBI2, LBI5, and LFI5). The primary structures of LPI5, LPI6, LBI5, LBI12 and LFI5 fractions are EPLYV, DPHI, AER, EQIDNLQ and WDDME, having IC50 values of 0.05, 0.02, 0.11, 0.24 and 0.01 g/L, respectively. Based on the results of simulated gastrointestinal enzyme degradation and ACE inhibitory activity, the <5Ka fractions of LPI6h, LBI2h and LFI2h have potential to be used in the preparation of functional foods.