Abstract: In order to clarify the physicochemical characteristics of the proteolytic digest of tuna cooking liquid and to investigate its osteoblast-promoting activity, the proteins in the tuna cooking liquid were separately hydrolysed by six proteases. The effects of different proteases on the proteolysis, molecular weight and amino acid composition of cooking liquids were investigated. The osteoblast-proliferative activities of different proteolyte was evaluated and the enzymatic process was optimized. Trypsin showed a high proteolysis (53.51%) of tuna cooking liquid protein. The proportion of small molecule peptides in the hydrolysate was 85.85% and was rich in glycine, tyrosine, phenylalanine that promoted osteoblast proliferation. Trypsin hydrolysate had the best osteoblast-proliferative activity, the dose-response relationship was consistent with y=0.4139x³−20.558x²+49.581x+99.203 (R²=0.9597). The optimal enzymatic process of trypsin was 3.5 h, 50 ℃, 10000 U/g specific activity of enzyme and pH8.0. Under the optimal process, the osteoblast proliferation rate of the tuna cooking liquid protein hydrolysate was 136.71%. Thus, trypsin has the potential to produce active peptides for osteoblast proliferation in tuna.