A selective enzyme-linked immunosorbent assay (ELISA) for the insecticide isofenphos was developed. Three different analogues (haptens) of isofenphos were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group t o use as immunogens or coating antigens. Rabbits were immunized with one of the haptens coupled to BSA for production of polyclonal antibodies and the sera were screened against each of the other two haptens coupled to ovalbumin (OVA). Using the sera of highest specificity, an antigen-coated ELISA was developed, which showed an I50 of 96 ng/mL with the detection limit of 2 ng/mL. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides and the phenol metabolite of isofenphos, which makes the developed assay suitable for the selective detection of isofenphos. An antibody-coated ELISA was also developed, which showed an I50 of 580 ng/mL with a detection limit of 70 ng/mL.