A method is described for the construction of a highly sensitive electrochemical biosensor for the detection of malathion, chlorpyrifos, monocrotophos and endosulfan based on covalent immobilization of acetylcholinesterase (AChE) on iron oxide nanoparticles (Fe(3)O(4)NPs)-decorated carboxylated multi-walled carbon nanotubes (c-MWCNTs) electrodeposited onto indium tin oxide (ITO)-coated glass plate. Transmission electron microscopic (TEM) and UV analysis of nanocomposite materials demonstrated that Fe(3)O(4)NPs were well deposited on the outer walls of c-MWCNTs. The modified electrode was characterized by atomic force microscopy (AFM), cyclic voltammetry (CV). Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The resulting biosensor exhibited a linear response for acetylthiocholine in a concentration range of 0.1-700 mu mol L-1 with a remarkable sensitivity of 0.402 mA/mu mol L-1. Under optimum conditions, the inhibition rates of pesticides were proportional to their concentrations in the range of 0.1-70 nmol L-1, 0.1-50 nmol L-1, 0.1-70 nmol L-1 and 0.1-100 nmol L-1 for malathion, chlorpyrifos, monocrotophos and endosulfan, respectively. The detection limit of the biosensor for all pesticides was 0.1 nmol L-1 at a signal-to-noise ratio of 3. The biosensor showed good reproducibility, no interference by metal ions and long-term stability. The measurement results obtained by the present biosensor were in good agreement with those obtained by the standard gas chromatography-mass spectrometry method. The biosensor was employed for the determination of pesticides in environmental and food samples. (C) 2012 Elsevier Ltd. All rights reserved.