Abstract: According to singe-factor experiments, three levels factors including the pH, temperature and salinity were selected to evaluate the optimum culture condition of Vibrio alginolyticus. Based on Box-Behnken central composite design principle of the Response Surface Methodology (RSM) , the OD600 was chosen as response value to determine the range of factors and levels. With the results, the optimized conditions of cultivation were as follows:pH7.62, temperature 34.55℃ and salinity 3.22%, respectively. During the germination significant interactions between pH and temperature, as well as temperature and salinity were found.