Objective To prepare and identify the monoclonal antibody against NPM1 protein. Methods The purified NPM1 fusion protein was used as the antigen. The hybrid cells were prepared by the conventional cell fusion technology. The hybrid cell line produced monoclonal antibody against NPM1 was screened by indirect ELISA. The characterizations of this antibody were analyzed. Results A hybrid cell line that produces monoclonal antibody against human NPM1 was screened. The subtype of antibody was IgG1 and the antibody titer reached 1:720000; the purity of monoclonal antibody was determined by SDS-PAGE and showed the two bands correspond to single heave chain and light chain of an antibody. The IHC results also showed this could detect the NPM1 expression on colorectal cancer. Conclusion The monoclonal antibody against NPM1 prepared in this study could be used for NPM1 protein function research and clinical examination study.