A radically new approach for the discriminative determination of various neurotoxins has been developed. This novel biosensor combines a highly sensitive acetylcholinesterase (AChE) biosensor with immobilized organophosphate hydrolase (OPH). The value of the new concept was demonstrated by the discrimination between carbamate and organophosphorus pesticides. It was shown that the response of traditional AChE-based biosensor to mixed samples containing paraoxon and carbofuran was not simply additive, and the measured concentrations of these pesticides were very different from their real concentrations. This combined OPH/AChE system was able to improve the accuracy of the AChE-based biosensor and to uniquely distinguish paraoxon in mixed solutions containing carbofuran. The presented approach promises a new perspective for “real world” analyses and opens a new area of discriminative determination of various species in multicomponent solutions.