In this research; enzymatic extraction of protamine peptides from primary tissues of sturgeon was studied. The optimum extraction technology was established; and the functional active protamine peptides were obtained. The results showed that the protamine hydrolysis of papain was significantly higher than that of pepsin and trypsin. On the basis of the single factor experiment with the conditions of adding amount; solid-liquid ratio; pH; the time and temperature of enzymatic hydrolysis; response surface methodology was further employed to establish a model for enzymatic hydrolysis of protamine peptides according to Box-Behnken design theory. The optimal extraction conditions were as follows: the solid-liquid ratio 39.72; papain 3%; pH 7.0 and temperature 55 ℃ for 8.22 h. The protamine hydrolysis degree reached to 5.44%. Moreover; the protamine peptides had good inhibitory effects onEscherichiacoliandStaphylococcus?aureus.