A simple, rapid, and sensitive method based on the acetylcholinesterase (AChE) reaction system and core-shell quantum dots (QDs) has been developed for the detection of organophosphorus pesticides and carbamates pesticides. The fluorescence of QDs could be effectively quenched by enzyme-generated H₂O₂, and the active centers of AChE could be inhibited in the presence of pesticides which causeed decrease of the generated H₂O₂. Then, the inhibition efficiency of pesticide to AChE activity could be evaluated by measuring the fluorescence changes. The important experimental conditions of the proposed biosensors were investigated and optimized. Under the optimized conditions, the lowest detection limit for selected analytes was 0.01 μM, which is much lower than the maximum residue limits (MRLs) regulated by the United States Food and Drug Administration (FDA) as well as European Union (EU). Moreover, the biosensor applications for the analysis of different kinds of pesticides in Panax ginseng were discussed based on the structure-activity relationship between pesticides and AChE. Featuring great selectivity, wide linear detection range, low detection limit and minimal sample size, the proposed method offers a rapid, sensitive, on-site and reliable method for routine pesticide detection in real sample, especially for the analytes with P=O structure in Panax ginseng.