In this paper, a novel acetylcholinesterase (AChE) biosensor was constructed by modifying glassy carbon electrode with CdTe quantum dots (QDs) and excellent conductive gold nanoparticles (GNPs) though chitosan microspheres to immobilize AChE. Since GNPs have shown widespread use particularly for constructing electrochemical biosensors through their high electron-transfer ability, the combined AChE exhibited high affinity to its substrate and thus a sensitive, fast and cheap method for determination of monocrotophos. The combination of CdTe QDs and GNPs promoted electron transfer and catalyzed the electro-oxidation of thiocholine, thus amplifying the detection sensitivity. This novel biosensing platform based on CdTe QDs-GNPs composite responded even more sensitively than that on CdTe QDs or GNPs alone because of the presence of synergistic effects in CdTe-GNPs film. The inhibition of monocrotophos was proportional to its concentration in two ranges, from 1 to 1000 ng mL(-1) and from 2 to 15 microg mL(-1), with a detection limit of 0.3 ng mL(-1). The proposed biosensor showed good precision and reproducibility, acceptable stability and accuracy in garlic samples analysis.