Nanostructured biosensors for the determination of organophosphorus pesticides (OPs) are fabricated by a layer-by-layer assembly technique. In the biosensors, bi-enzymes of acetylcholinesterase (AChE) and choline oxidase (ChOx) are used as biological receptors, while CdTe quantum dots (QDs) are explored as fluorescent probes for optical transduction of the enzymatic activity. Increasing amounts of OPs lead to a decrease of the enzymatic activity and thus a decrease in the production of hydrogen peroxide, which can quench the fluorescence of the CdTe QDs. The decrease of quenching rate is relative to the concentration of OPs. Using this biosensor, monitoring of three types of commonly used OPs (paraoxon, dichlorvos and parathion) at picomolar levels is realized. The linear range of detection covers six orders of magnitude (10−12 to 10−6 M). In addition, the biosensors exhibit a similar limit of detection and calibration curves for these pesticides, which allow them to be used for the accurate determination of total OPs and carbamate content (not the sum of anti-acetylcholinesterase toxicity as obtained by standard cholinesterase inhibition assay) of mixtures of OPs and carbamate pesticides. Finally, detection of OPs in fruits at very low levels has been achieved.