A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed to measure anilofos levels in agricultural and environmental samples. The ELISA was developed using rabbit polyclonal antibodies against a hapten-protein conjugate of anilofos-bovine serum albumin. The limit of detection was 0.1 μg L(-1), and there was no cross-reactivity with other related pesticides or structurally similar compounds. The matrix effects of rice (aromatic rice, white rice, brown rice), corn, barley, wheat and soil were measured and removed by extraction and dilution with phosphate buffered saline with 0.05% Tween-20. For water samples (tap water and river water), the matrix effects were also removed by dilution with phosphate buffered saline with Tween-20. The detection limits for anilofos in authentic samples (aromatic rice, white rice, brown rice, corn, barley, wheat, soil, tap water and river water) were 2, 2, 2, 3, 2, 2, and 2 μg kg(-1), and 0.5 and 1 μg L(-1), respectively . The anilofos recovery ranged from 81.0-116.0% with a coefficient of variation of 1.7-9.0%. The method was validated using GC, and the results showed good correlation with the dc-ELISA data (r(2) = 0.9795). Forty-two cereal samples were randomly collected from different supermarkets and analyzed using the developed dc-ELISA. No anilofos was found in these products. The developed immunoassay is suitable for rapid quantitation of anilofos residues.