The invention discloses a monoclonal antibody of anti-human calnexin protein, a preparation method and the application thereof, belonging to the preparation of antibody-containing medical preparation or the monoclonal antibody. The monoclonal antibody of the anti-human calnexin protein is generated by a hybridoma cell with the collection number of CGMCC No. 3240. The breast adenocarcinoma MCF-7 immuno-BALB/c mouse is adopted, spleen and myeloma cell of the mouse are taken to make cell fusion, the MCF-7 cell is utilized for screening, and then a monoclonal antibody library is formed by the obtained positive clones; and the antigen of the monoclonal antibody of the anti-human calnexin protein derived from the antibody library can be identified to be calnexin by immuno-precipitation and a mass spectrum method. The antibody and the calnexin protein have specific binding capacity, and a variable region sequence for identifying the antibody can be determined. The invention is applicable to the detection of immunohistochemistry, immuno-fluorescence, immuno-blot and immuno-precipitation of the molecule.