This work describes the fundamental ability of a commercial ELISA to determine acetamiprid and the application of the ELISA to residue analysis in fruit and vegetable samples. The ELISA exhibited satisfactory sensitivity (I (50) 0.6 ng/g; limit of detection 0.053 ng/g) and a high selectivity for acetamiprid versus other neonicotinoid analogs (thiacloprid amide). Methanol, which influenced the sensitivity of the ELISA the least, was selected as the extractant for the ELISA analysis. Simple dilution of sample extracts with water eliminated matrix interferences. Average recoveries from the acetamiprid-spiked agricultural samples were >95% using a simple extraction method. Analytical results obtained from the ELISA were comparable to those obtained from the reference HPLC method (r>0.99). The ELISA applied to the residue analysis of acetamiprid in agricultural products is a rapid, simple, and cost-effective method, and could be successfully applied to the detection of acetamiprid before the distribution of produce.