Herein, a facile electrochemical acetylcholinesterase (EC 3.1.1.7; AChE) biosensor based on nafion (NA) and Ag nanoparticles supported on amine functionalized reduced graphene oxide (rGO-NH2) was developed. The Ag@rGO-NH2 nanocomposite was characterized using Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), and X-ray diffraction (XRD). After being optimized, the biosensor exhibited excellent electrochemical response to the oxidation of thiocholine, the hydrolysis product of acetylthiocholine chloride (ATCl) catalyzed by AChE. An apparent Michealis-Menten value of 20.5 mu M was obtained. Under optimized conductions, the biosensor detected malation, methidathion, and chlorpyrifos ethyl in the linear range from 0.0063 to 0.077 mu g/mL, from 0.012 to 0.105 mu g/mL, and from 0.021 to 0.122 mu g/mL, respectively. The detection limit (LoD) was 4.5 ng/mL for malation, 9.5 ng/mL for methidathion, and 14 ng/mL for chlorpyrifos ethyl. Also, the NA/Ag@rGO-NH2/ AChE/GCE biosensor showed god sensitivity, stability and repeatability, which provides a promising tool for the detection of organophosphate pesticides. (C) 2017 Elsevier Ltd. All rights reserved.