Abstract: To study the comprehensive utilization of by-products from eel processing, the angiotensin-converting enzyme inhibitory peptide was prepared from eel (Anguilla japonica) bone collagen by enzymatic hydrolysis. The ACE inhibitory activity and degree of hydrolysis were used as evaluation indexes, we determined the optimal hydrolysis conditions by single factor and response surface experiments. Furthermore, the amino acid composition and molecular mass distribution of the hydrolysate prepared under the optimized conditions were determined. The results show that alkaline protease was the optimal enzyme, and the optimal hydrolysis conditions were as follows: temperature of 50 ℃, mass concentration of 15 g·L⁻¹, hydrolysis time of 5.25 h, enzyme dosage of 3.1% (Mass fraction) and pH of 9.2. Under these conditions, the ACE inhibitory activity was 70.33%, which was close to the predicted value. The molecular weight of peptides below 1 000 D and 1 000–3 000 D in enzymatic hydrolysate accounted for 57.02% and 36.55%, respectively. Amino acid composition analysis shows that the content of hydrophobic amino acids related to ACE inhibition activities (Such as Pro、Val、Ile、Leu、Phe) increased.