Abstract: In order to enhance the flavor of hydrolysate from the mixture of chicken skeleton and chicken breast, the hydrolysis of chicken skeleton and chicken breast composite substrate was carried out by biological enzymatic hydrolysis technology. The enzymatic hydrolysis process was optimized by single factor experiment and orthogonal experiment. Furthermore, the free amino acid content and molecular weight distribution of peptide in hydrolysates were analyzed. The results pointed out that the flavor of hydrolysate was better using simultaneous hydrolysis with protamex and flavourzyme by the ratio of 3:1. The optimal enzymolysis conditions were as follows: Ratio of chicken skeleton to chicken breast 9:1, solid-liquid ratio 1:2 g/mL, temperature 50 ℃, pH7.5, enzyme dosage 0.6%, and hydrolysis time 3 h. Under these conditions, the hydrolysate had a prominent umami taste, obvious meat flavor and good taste, with hydrolysis degree and sensory score of 13.60% and 4.7, respectively. With the prolongation of enzymatic hydrolysis time, the content of total free amino acids and peptides with low molecular weight increased continuously. Under the optimal process conditions, the content of bitter amino acids in the hydrolysate was 2.74 mg/mL, accounting for 71.73% of the total free amino acids, which were the main amino acids, and the content of peptides with molecular weight less than 3 kDa was 93.26%. The results provide a reference for the comprehensive development and application of low-value chicken skeleton.