Abstract Neurotoxic organophosphorus (OP) compounds are commonly used as chemical warfare agents and pesticides. Due to their high toxicity, rapid and sensitive field detection of these compounds has been an ongoing topic of interest. Biosensors made with organophosphate hydrolase enzyme (OPH) are generally designed to either amperometrically detect an electroactive leaving group produced following enzymatic cleavage, or to potenitometrically detect the pH change that occurs during cleavage. Since OPs are more likely to have phenolic leaving groups as compared to electroactive leaving groups, we have developed a new amperometric dual enzyme electrochemical assay that enables the detection of a broad class of OP compounds using the OPH enzyme combined with horseradish peroxidase (HRP). The assay has been applied to the detection of dichlofenthion, which does not have an electroactive leaving group and is not a commonly investigated OPH substrate. Using reverse phase HPLC, we have determined the Michaelis–Menten kinetic parameters of an engineered OPH enzyme to be K M = 0.11 ± 0.02 mM and k cat = 0.046 ± 0.003 s −1 with dichlofenthion as the substrate. Detection of the phenolic leaving group from the OPH enzyme reaction using the HRP electrode is carried out at −50 mV vs. Ag/AgCl where the noise and background are low and interferences are negligible. After optimization of the solution pH, the dual enzyme biosensor was found to have a limit of detection (LOD) of 24 μM (7.6 ppm), and a sensitivity of 0.095 ± 0.024 nA/μM for dichlorofenthion. By detecting the phenolic leaving groups from the OP targets using the HRP electrode, biosensors made using this new platform have the potential to detect a broad range of important OP compounds.