To realize the high comprehensive utilization on the byproduct of silver carp, taking the testis sacs of silver carp as the raw material, the effects were studied, including the concentration of citric acid, the temperature, ultrasonic power, ultrasonic time, solid-liquid rate and extraction times on the efficacy extraction of silver carp spermary. On the base of single-factor experiments, and through the orthogonal test, the optimization experiment conditions were as followed: concentration of citric acid 0.45 mol/L, extraction temperature 55 ℃, the ultrasonic time 20 min , ultrasonic power 400 W, ratio of solid-liquid 130, and the extraction times 3 times. The extraction ratio of chub protamine was 3.25% at the optimized conditions. Four groups chub protamine were purified and separated through sepharose fast flow ion-exchanged chromatography. By studying the antibacterial activities of the four group, excepting the precursor protein, the other groups had a good inhibition on the salmonella, staphylococcus aureus and escherichia coli, especially the group II and III.