Abstract: Alkaline protease, pepsin and trypsin were used to hydrolyze the bones and meat, shell and skirt of Chinese soft-shelled turtle to prepare functional peptides, and their physical and chemical properties such as main conformational changes and particle size were analyzed. The potential bone mineral density-regulating activity of each enzymatic hydrolyzate was evaluated by measuring the effect on proliferation of MC3T3-E1 cells. The results showed that the protein content of Chinese soft-shelled turtle shell and skirt dry powder was 54.18%, and the protein content of bone and meat dry powder was 80.38%. After 2 h of enzymatic hydrolysis, the molecular weights of alkaline protease and trypsin hydrolyzed products were concentrated below 1000 Da, and the molecular weight distribution of pepsin hydrolyzed products was relatively uniform. The structure of the enzymatic hydrolysis products was dominated by random coil, and the particle sizes were concentrated in the 20 and 200 nm regions, indicating that the enzymatic hydrolysis was sufficient. The content of essential amino acids such as lysine, valine, phenylalanine, leucine and threonine in the enzymatic hydrolysis products was relatively high, and the content of glycine, alanine and proline in non-essential amino acids was relatively high. Among them, the total amount of essential amino acids after enzymatic hydrolysis of Chinese soft-shelled turtle bones and meat by pepsin reached 39.81%. The results of cell experiments showed that the six enzymatic hydrolysates had a certain pro-osteogenic activity, and the proliferative activities of the enzymatic hydrolysates of shell and skirt were stronger than those of bone meat, and the activity of the shell and skirt products of pepsin enzymatic hydrolysis was the best. The above data showed that the enzymatic hydrolysate of Chinese soft-shelled turtle had potential bone density regulation activity. This research provides feasibility analysis and theoretical reference for deep processing of Chinese soft-shelled turtle products.