Abstract: [Abstract] Objective：This study aimed to prepare monoclonal antibodies against human amelogenin and characterize their immunological properties. Methods：BALB/c mice were immunized with the recombinant full-length human amelogenin expressed in prokaryotic expression system. The spleen cells of immunized mice were fused with mouse myeloma SP2/0 cells, and hybridoma cell lines capable of stably secreting monoclonal antibodies were screened out by the limited dilution method and indirect enzyme-linked immunosorbent assay (ELISA) method. Ascites were produced in mice, and then purified by saturated ammonium sulfate precipitation and protein G affinity chromatography. Results：Three hybridoma cell lines stably secreting monoclonal antibodies against human amelogenin were screened and named as H10G1, H3G1 and G5H3. The ascites titers were 1:243000, 1:729000, 1:729000, and the titers of monoclonal antibody after purification was 1:81000、1:243000、1:729000, respectively. The results of Western blotting showed that all of 3 monoclonal antibodies recognize the 45-149 amino acid of human amelogenin. Conclusions：The specific monoclonal antibody against human amelogenin was successfully prepared for further study on the amelogenin in root development and periodontal regeneration.