The invention provides a nematode-trapping fungus lyophilized preparation as well as a preparation method and application thereof. The nematode-trapping fungus lyophilized preparation is prepared by the following steps: carrying out chlamydospore large-batch culture on nematode-trapping fungus isolated strains, eluting with 0.04%-0.06% sterilization Tween-80 and filtering to prepare a 0.3-0.6*10<6>/ml spore solution; carrying out elution protection with 10%-20% beta-cyclodextrine; and finally, pre-freezing, freezing and drying in vacuum to prepare the product. The invention further provides a nematode-trapping fungus oral biological preparation as well as a preparation method and application thereof. The nematode-trapping fungus lyophilized preparation provided by the invention can be orally taken, also can keep good activity in links of storing, transporting and the like, and has a very good protection effect on chlamydospores; and the cost is saved and the implementation is convenient. The preparation method provided by the invention is suitable for storing strains, and is relatively suitable for producing lyophilized preparations in a large scale.