The invention discloses a separation and cultivation method for isaria fumosorosea. The method comprises the following steps of collecting bemisia tabaci bodies infected with entomogenous fungus from vegetable leaves, separating the entomogenous fungus from the bodies, putting the entomogenous fungus on a PDA (potato dextrose agar) culture medium for cultivating to obtain spore suspension which is sprayed on two-year-old nymphs of bemisia tabaci, then separating the entomogenous fungus from the bodies again after cultivation, storing the entomogenous fungus on a slope of a test tube and storing the test tube in a refrigerator; taking the entomogenous fungus out of the refrigerator, preparing the spore suspension, dipping the spore suspension through filtration paper, inoculating the spore suspension onto the culture medium, cultivating in a mildew incubator, picking up hypha, inoculating the hypha into germfree water, then stirring and filtering; putting the spore suspension at the center of a culture dish, perforating a hole in the center of a bacterial colony, dispersing tween-80, stirring and filtering again, and calculating the sporulation quantity and the diameter of the bacterial colony. The method is used for screening an optimal culture medium formula, the optimal temperature, an optimal pH, a lighting condition and a ventilating condition. The method has the advantages that the diameter area of the bacterial colony is large and the sporulation yield is large.