Abstract: Objective: Prepare monoclonal antibodies ( Mc Abs) against copper, and establish indirect and direct competitive enzyme-linked immunoassay for detection of copper. Finally rapid quantitative determination of Cu2 +was achieved. Methods:Using double functional chelating agent p-SCN-Bn-NOTA to connect Cu2 +with carrier protein BSA and OVA, Immune Balb/c mice.After cell fusion, positive cells that stability secrete Mc Abs against Cu2 +were selected through indirect and indirect competitive ELISA. Preparation and identification of Mc Abs. Established indirect and direct competitive ELISA detection method.Results: 4 hybridoma cell lines that stability secreting Mc Abs against Cu2 +were prepared successfully, the titer of G8 was 5 × 105.The subtype and type of light chain were Ig G1 and κ, specific detection showed that the cross-reactivity of Mc Ab with Mn2 +, Pb2 +, Cd2 +, Zn2 +and NOTA were less than 2%, except for Mg2 +which was less than 8%, this showed high specificity of this antibody. The detection sensitivity of established indirect competitive ELISA was 29.8 ng/m L, Detection of limit was 2.4 ng/m L and the detection rang was from 4.5 to 196.7 ng/m L. The detection sensitivity of established direct competitive ELISA was 12.89 ng/m L, detection of limit was 0.88 ng/m L and the detection rang was from 1.72 to 96.58 ng/m L.Conclusion: Successfully prepared Mc Abs against Cu2 +and established indirect competitive ELISA and direct competitive ELISA.