The environmental pollution by heavy metals such as mercury, cadmium and lead has become a worldwide public health hazard. To rapidly and inexpensively monitor environmental heavy metals is a prerequisite for minimizing human and animal exposure. The development of immunoassays to detect mercury ion residues has been a promising trend with the advantage of rapid and cheap operation. We reported the isolation and characterization of mercury-specific monoclonal antibodies. Because Hg2+ ions are too small to elicit an immune response, the metal was coupled to protein carrier (keyhole limpet, KLH) using a chelator (diethylenetriamine pentaacetic acid, DTPA). After the synthesis of antigen and characterization, monoclonal antibodies against mercury ions were generated by immunizing BALB/c mice with mercury conjugated antigen (Hg-DTPA-KLH). The stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma cells. The hybridoma cells were subcloned by the limiting dilution and screened by ELISA, two hybridoma cell lines producing stably specific monoclonal antibodies (MAbs) against mercury ions were obtained, named H2H5 and H1H8. The ascites fluid was produced in BABL/c mice by intraperitoneal injection of 1×107 H2H5 and H1H8 cells, respectively. The titers of ascites were all above 1:51 200. The isotyping of secrete antibodies from two hybridoma cell lines was IgG1, kappa type. These data laid a potency of establishing immunoassays methods of determining Hg2+ ion residues and had the realistic significance for improving the efficiency and quality of risk assessment.