To produce high-affinity monoclonal antibodies against pesticide imidacloprid, we synthesized the haptens 1-[(6-Carboxylethylthio-3-pyridinyl) methyl] -N-nitro-imidazolidinimine (named as H1) and 1-[(6-Chloro-3-pyridinyl) methyl]-3-carboxylpropyl-N-nitro-2-imidazolidinimine (termed as H2). And then the haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA) for immunogen (H1-BSA) and coating antigen (H2-OVA) respectively by NHS ester method. BALB/c mice were immunized with H1-BSA conjugate. We obtained two hybridoma cell lines 2F11/A9 and 2G6/G12 secreting antibody specific for imidacloprid from the conventional hybridoma technology. The result showed that the subtypes of obtained monoclonal antibodies were IgG3 and IgG1, respectively, and the titers of ascites were up to 1:128 000. The indirect competitive ELISA indicated the IC50 values of 5.3 and 28.3 ng/mL with detection limits of 1.1 ng/mL and 7.7 ng/mL, respectively. Two monoclonal antibodies had no apparent cross reactivity with six analogous compounds. Thus, two prepared monoclonal antibodies had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of imidacloprid residue and laid a solid foundation for research and development of products for immunoassay.