The protein extraction ratio of DTSM could reach to more than 85.64% when the DTSM with particle sizes of 0.25 mm was hydrolyzed by papain for 5 h. The results proved that the enzymatic hydrolysis is a promising method to extract protein from DTSM. The optimization results showed that the optimal hydrolysis condition for preparing the highest concentration of glutamic acid and aspartic acid were enzyme activity of 18.22%; pH of 3; hydrolysis temperature of 40 C; and hydrolysis time of 6 h, and the corresponding concentration of the glutamic acid and aspartic acid obtained were 727.6 mg/mL and 149.9 mg/mL, respectively. The pH and enzyme activity were the most important factors affecting the hydrolysis of DTSM using papain, which need to be monitored for during papain hydrolysis of DTSM in large scale applications.