Abstract: Objective: To determine the optimum process condition for enzymatic hydrolysis of Penis et Testis Cervi peptides by alcalase, and to explore its activity of tonifying kidney and strengthing bone in vitro. Methods: With the temperature, pH, time and dosage of enzyme as the influencing factors, and the degree of hydrolysis as the response index, the response surface test was designed based on the single factor experiments and Box-Behnken method to obtain the optimal enzymatic hydrolysis condition of Penis et Testis Cervi peptides. To investigate the effects of different concentrations of Penis et Testis Cervi peptide on the survival rate of TM3 cells and the secretion of testosterone (T), and on the survival rate of MC3T3-E1 cells, the activity of alkaline phosphatase (ALP), and the secretion of osteocalcin (OCN). Results: The optimal hydrolysis condition was temperature 51 ℃, pH8.7, time 4.1 h, enzyme dosage 1300 U/g, and the degree of hydrolysis was 40.36%±0.22%. The results of in vitro activity experiments showed that different concentrations (25, 50, 100, and 200 μg/mL) of TM3 cells compared with the model group could significantly increase cell viability and T secretion (P<0.05). At the mass concentration of 50 μg/mL, the T secretion of TM3 cells was most promoted by the enzymolysis peptide from Penis et Testis Cervi, with the secretion amount of 42.47 ng/mL. Compared with the model group, the cell viability, ALP activity and OCN secretion were significantly increased in the MC3T3-E1 cell medicated groups at different concentrations (25, 50, 100, and 200 g/mL) (P<0.05). At the mass concentration of 50 μg/mL, the activities of ALP and OCN secretion were the most significant, with ALP activity of 6.80 King/g prot and OCN secretion of 15.63 ng/mL. Conclusion: The response surface methodology was used to determine the optimal enzymatic hydrolysis process of Penis et Testis Cervi peptide, which verified that the enzymolysis peptide had good activity of tonifying kidney and strengthing bone in vitro, and would provide a theoretical basis for the comprehensive development and utilization of Penis et Testis Cervi in the future.