The invention discloses a microbial inoculum for preparing a caged chicken fermentation mattress material and a preparation method of the inoculum. The composite microbial inoculum is prepared by inoculating a composition consisting of a bacillus amyloliquefaciens fermentation liquid, a bacillus megaterium fermentation liquid, a trichoderma longibrachiatum fermentation liquid and a lactobacillus plantarum fermentation liquid into a solid culture medium, uniformly mixing, culturing for 72-96 hours under a culture condition of 30-35 DEG C, and drying. The invention discloses an application of the composite microbial inoculum in preparing the caged chicken fermentation mattress material. The composite microbial inoculum is prepared by selecting the best proportion of four excellent strains, the functional strains cooperate in the feeding and management process of the caged chicken fermentation bed surface, the functions of the organic matter transforming bacteria, the ammonia fixing deodorizing bacteria and the bacteria inhibiting bacteria are complementary, the temperature rise fermentation of the fermentation bed is rapidly started, the rapid removal of moisture in feces is realized, the microbial diversity of the fermentation bed is improved, the growth of pathogenic spoilage bacteria is inhibited, the in-situ degradation of chicken manure is promoted, and the humification rateof the mattress material is improved.