Abstract: Experiments of extracting protamine with ultrasound assistant processing in bovine testes were designed using Box-Behnken response surface methodology. The optimal extraction processing parameters based on design expert were:5.5% sulfuric acid, 190 W of ultrasonic and extraction time was 10 min. Compared with the traditional extraction method, this study raised the protamine productivity up to 33.87%, decreased the extraction time to 10 min and increased the content of protamine with up to 6.47μg/g as well. In addition, while the optimal precipitating reagent was 95% ethanol, volumeration 1∶3 for testicular precipitation, protamine product yield, water soluble and color were the best.