A novel and sensitive enhanced chemiluminescence enzyme-linked immunosorbent assay (ECL-ELISA) for the simultaneous analysis of imidaclothiz and thiacloprid is described. The concentrations of coating antibodies and enzyme tracers were optimised by three different ECL-ELISA formats. Under the optimised conditions, when anti-imidaclothiz polyclonal and anti-thiacloprid polyclonal antibodies were immobilised in a single well, single-enzyme tracer ECL-ELISA was used to screen the corresponding pesticide residue, and multi-enzyme tracer ECL-ELISA was used for the analysis of imidaclothiz and thiacloprid residue mixtures. The average recoveries of the two pesticides from spiked tomato, cabbage, and rice samples were in the range of 83.7–117%. Meanwhile, the results showed that the multi-enzyme tracer ECL-ELISA could be applied to the accurate analysis of different proportions of imidaclothiz and thiacloprid (1 : 4 to 4 : 1, m/m) when the gross residues ranged from 20 to 250 μg kg−1. The results of the multi-enzyme tracer ECL-ELISA in the real tomato samples correlated well with those of high-performance liquid chromatography, with a correlation coefficient of 0.996. Therefore, this new strategy for developing immunoassays is suitable for the simultaneous quantitative detection of imidaclothiz and thiacloprid residues in agricultural samples.
