Objective: The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initially in clinical immunoassay. Methods: Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by affinity chromatography. Antibody titer was determined by enzyme linked immunosorbent assay(ELISA). The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results: Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by affinity chromatography. The purity of antibodies was higher than90%. The titers of antibodies were more than 1: 6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor cell lines and determine the expression of P53 in tumor tissue. Conclusion: Three strains of monoclonal antibodies with high affinity to P53 were successfully established, which can be used for detecting the expression of P53 in tumor cells or tissue. Objective: The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein, and use the antibody initially in clinical immunoassay. Methods: Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results: Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by affinity chromatography. The purity of antibodies was higher than 90%. The titers of antibodies were more than 1: 6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor cell lines and determine the expression of P53 in tumor tissue. Conclusion: Three strains of monoclonal a ntibodies with high affinity to P53 were successfully established, which can be used for detecting the expression of P53 in tumor cells or tissue.