A competitive enzyme immunoassay for the quantitation of chlorpyrifos in water that utilizes amine-terminated superparamagnetic particles as the solid phase to covalently attach mouse monoclonal anti-chlorpyrifos antibodies was developed. Specificity studies indicate that the monoclonal antibody can distinguish chlorpyrifos from its major degradation products, including 3,5,6-trichloro-2-pyridinol, and structurally similar pesticides. The immunoassay has an estimated limit of detection of 0.10 ng/mL in water and compares favorably with traditional chromatographic methods when water samples are analyzed by both methods (r = 0.985). Within- and between-assay percent coefficient of variation values are <13%, and recovery of chlorpyrifos from water averaged 97% across the range of the immunoassay method.