In this study, Sephadex G-50 and CM Sepharose Fast Flow were used to purify the spermatozoa of Takifugu obscurus. Identification of protamine by arginine color reaction. The effects of protamine on the minimum inhibitory concentration, inhibition zone diameter and growth curve of Staphylococcus aureus, Escherichia coli and Salmonella were determined. The results showed that: The spermatozoa of Takifugu obscurus had an absorption peak at 217 and 273 nm, respectively, and the absorption peak at 217 nm was higher. The purified protamine was found to be better than the protamine before purification by SDS-PAGE electrophoresis. The strips were fine and had no tailing. The Takifugu obscurus protamine had inhibitory effects on Gram-negative bacteria (Salmonella and Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus), which could delay the bacteria growth, and the minimum inhibitory concentrations against Staphylococcus aureus, Escherichia coli and Salmonella were 500, 1 000 and 1 000 mg/L, respectively.