Abstract: In this study,tuna skin was prepared by enzymatic hydrolysis after ultrasonic pretreatment to produce hydrolysate with ACE inhibitory activity. This hydrolysate was used as raw material,the gastrointestinal digestion in vitro of hydrolysate was tested. The results showed that all peptides from tuna skin keep high ACE inhibitory activity after digested. After 20 min and 40 min of ultrasonic pretreatment,the ACE inhibitory activity of digested products by enzymatic hydrolysis for 5 min was significantly higher than that of other groups(p<0.05). E5U20 and E5U40 was separated into three components by Sephadex G-15,the peak F2 of E5U20 digestible products was best,of which IC₅₀ value was 0.393 mg/mL. And then,the F2 was further separated by semi-high performance liquid chromatographic. There were 8 peaks after separated,peak 3 had a lowest IC₅₀ of 0.102 mg/mL.Finally,the peak 3 was identified by mass spectrometry,the results showed that peak 3 peptide sequence was GPSGPPGP,this sequence was derived from 842~849 of collagen α1 chain. And these amino acids were consistent with the structure-activity characteristics of ACE inhibitory peptides,and the sequences were not reported by now.