To develop a competitive indirect enzyme-linked immunosorbent assay for metamifop, a new aryloxyphenoxypropionic acid herbicide, three structurally related haptens were synthesized. Hapten conjugates to keyhole limpet hemocyanin and bovine serum albumin were used as immunogens and plate-coating antigens, respectively. Various sets of polyclonal antibodies from rabbits and the coating antigens were screened for the assay in simple homologous and heterologous ELISA formats. A selected heterologous ELISA was optimized to show an average IC50 value as low as 20.1 ng/mL, detection ranges of 1.0-350 ng/mL, and a lowest detection limit of 0.1 ng/mL. The cross-reactivities of other aryloxyphenoxypropionic acid herbicides to the antibodies were less than 0.5% in the assays except fenoxaprop-P and fenoxaprop-P ethyl, having a diaryl ether group identical to that of metamifop. Molecular modeling studies revealed that the physicochemical properties of the diaryl ether group are the most important determinants of sensitivity and selectivity. The results strongly indicate that the selected set of ELISA is a highly sensitive and convenient tool for detecting metamifop.