The invention discloses an anti-rabies virus monoclonal antibody and a preparation method and an application, belonging to the field of biomedicine and particularly relating to the preparation of a monoclonal antibody capable of identifying rabies virus and the application. The monoclonal antibody of the invention is screened by indirect Enzyme-linked immunosorbent assay (ELISA), and specificity and affinity thereof combined with antigen are identified by methods such as polyacrylamide gel electrophoresis analysis, speckle ELISA, immunoblot analysis and the like. The anti-rabies virus monoclonal antibody of the invention can be applied in multiple testing methods of antigen of rabies virus and can be also applied in the preparation of rabies virus detecting kit. The anti-rabies virus monoclonal antibody is secreted by anti-rabies virus monoclonal antibody hybridoma cell strain 2C5 with the preservation number being CGMCC No.3014.