Abstract: In order to obtain a proper isolation medium for B. bassiana, through the colony number on the medium of B. bassiana and other bacterial with different concentration of three kinds bacteriostatic agents, to identify which ratio was reasonable that could mix the bacteria and grow better for B. bassiana. The result showed that, the PPDA medium with 70 mg/L dodine, 125 mg/L cephalothin and 138 mg/L kanamycin sulfate was the best. The amount of B. bassiana 30 days after application of two formulation B. bassiana pesticide was detected by this new isolation medium, the data showed that the suspension of B. bassiana were more persistent than granules in field significantly. It was proved that this new selective isolation medium was suitable for application.