Abstract: In this study, cod bones were used as raw material. The bitterness of cod bone hydrolysate was reduced by controlling the degree of hydrolysis, and the mechanism of peptide and free amino acids in the hydrolysate on bitterness was analyzed. The results showed that the degree of hydrolysis reached 7.48% and the bitterness value was 5.80 through mild hydrolysis for 2 h. The peptides were mainly distributed between 1500 and 2000 Da. In addition, the precipitate was subjected to high-pressure cooking and heat treatment (121 °C, 30 min) to make the surface of the fish bone loose after the first enzymolysis. The degree of hydrolysis reached 49.24% and the bitterness value was 6.03 through extensive hydrolysis for 2 h. The peptides were mainly distributed between 500 and 1000 Da. Free amino acids were significantly higher than the content after only the first hydrolysis. By controlling the degree of hydrolysis to limit the production of bitterness, the preparation of this low-bitterness enzymatic peptide laid the foundation for its application in food.