Abstract: The extraction and separation of protamine from sturgeon by H2SO4 was studied and the optimal extraction process was set up.Taking the protein yield as the main indicator,the optimal separating conditions were initial determined on the basis of single factor tests on the influence of the concentration and amount of H2SO4,extraction temperature,extraction duration and extraction times.The best extraction conditions were finally confirmed with the Box- Behnken design in Design- Expert software:the concentration of H2SO4 was 0.83 mol / L while the amount was 6.2∶ 1 times volume,the extraction temperature was better at35 ℃ and extracting 0.5 h for 4 times was recommended. It proved that the mean yield of protamine was 11.28% under the optimal extraction conditions.Moreover,the extracted protamine has been shown to have not only high purity of 86.13% and low molecular weight of 10 ku but also good antibacterial activity against staphylococcus aureus with the inhibition zone diameter of15.75 mm.