The invention discloses a culture medium for cultivating high-toxicity Metarhizium anisopliae and a preparation of the Metarhizium anisopliae and application of the preparation, and belong to the technical field of plant protection. The culture medium comprises the following components: wheat bran, rice husks, rice flour, corn flour, polypide lyophilized powder, dipotassium hydrogen phosphate, manganese sulfate, calcium chloride and water. Since the polypide lyophilized powder of Spodoptera frugiperda is introduced into a traditional culture medium, the toxicity of Metarhizium anisopliae NG-Ma040 for Spodoptera frugiperda killing can be improved, and no obvious influences on the sporulation quantity can be caused; meanwhile, through synergistic cooperation among the wheat bran, rice husks,rice flour, corn flour, dipotassium hydrogen phosphate, manganese sulfate, calcium chloride and other components, the toxicity for Spodoptera frugiperda is improved further, and the infection rate and control effect on larvae of Spodoptera frugiperda can be improved; and moreover, the culture medium can be made into the preparation of Metarhizium anisopliae, and the preparation is applied to prevention and control of Spodoptera frugiperda.