The invention belongs to the field of immunodetection, and particularly discloses three kits for detecting human phosphorylated Tau protein and a preparation method thereof. According to the three detection kits disclosed by the invention, the monoclonal antibody 3G5 is used as a capture antibody, and the monoclonal antibody 4B1 is used as a detection antibody, or the monoclonal antibody 4B1 is used as a capture antibody, and the monoclonal antibody 3G5 is used as a detection antibody, wherein p-hTau396, 404 protein can be detected by the kit. The three detection kits constructed by using the monoclonal antibodies 3G5 and 4B1 are respectively suitable for detection in different scenes. The method can rapidly and accurately detect the human phosphorylated Tau, integrates the characteristics of specificity of the antibody, high sensitivity of ELISA, efficient enrichment of the immune nano magnetic beads, convenience and rapidness of the colloidal gold test strip and the like, and has the characteristics of accuracy, rapidness, high efficiency, specificity and sensitivity. Therefore, the method is suitable for rapid and sensitive early diagnosis of Alzheimer's disease, and is of great significance to timely and accurate guidance of medicine use for patients.