Mercury is one of the most toxic heavy metals present in the environment. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the determination of Hg(2+) was developed. A new bifunctional ligand, 6-mercaptonicotinic acid (MNA), which contains a pyridine ring bearing a carboxylic group and a mercapto group, was selected for the preparation of immunogen. After immunization of mice and performing the hybridoma technique, the obtained mAb was characterized for its binding affinity and selectivity for Hg(2+). Based on this novel mAb, an ELISA was established. At optimal experimental conditions, the standard curve of the ELISA for Hg(2+) was constructed in concentration range of 0.1-100 ng mL(-1). The values of IC(50) and LOD of the assay were found to be 1.12 and 0.08 ng mL(-1). The cross-reactivity was lower than 2% with MNA, CH(3)Hg, and CH(3)Hg-MNA and was 11.5% and 4.4% for Hg(+) and Au(3+), respectively. No cross-reactivity was found with other metal ions such as Cu(2+), Sn(2+), Ni(2+), Mn(2+), Pb(2+), Zn(2+), Cd(2+), Fe(2+), Co(2+), Mg(2+), Ca(2+), and anions such as Cl(-), NO(3)(-), NO(2)(-), HCO(3)(-), F(-), and SO(4)(2-), indicating that the assay displays not only high sensitivity but also high selectivity. Different kinds of samples including water, milk, green vegetable, kelp, facial cleanser, and night cream were spiked with Hg(2+) and the extracts were analyzed by ELISA. Acceptable recovery rates of 80.0-113.0% and coefficients of variation of 1.9-18.6% were obtained. A linear relationship between ELISA and cold-vapor atomic fluorescence spectroscopy (CV-AFS) as indicated by a correlation coefficient of 0.97 for liquid samples (water samples) and 0.98 for other samples was obtained. The proposed mAb-based ELISA provides a feasible analytical method for highly sensitive and specific, fast, simple, and accurate determination of uncomplexed trace Hg(2+) in environmental and food samples.