The invention discloses a phosphorus removal microbial inoculant and a preparation method and application thereof. The microbial inoculant contains pseudomonas and streptomycete. The pseudomonas is pseudomonas W12, which is preserved in China General Microbiological Culture Collection Center on May 17, 2016, and has a preservation number of CGMCCNo.12460. The streptomycete is streptomycete GS14, which is preserved in China General Microbiological Culture Collection Center on January 8, 2013, and has the preservation number CGMCCNo.7095. The microbial inoculant is prepared by fermenting and culturing activated pseudomonas or streptomycete, and mixing. The phosphorus removal microbial inoculant can be used for enhancing biological phosphorus removal of household sewage. The present invention provides two novel phosphorus microbes: pseudomonas W12 and streptomycete GS14. The phosphorus removal microbial inoculant can be used in sewage treatment, not only can remove the total content of phosphorus in sewage, but also has good effect on removal of total nitrogen.