A nanocomposite consisting of conducting polymer (CP)- Poly(3,4-ethylenedioxythiophene) (PEDOT) and multi-walled carbon nanotubes (MWCNTs) has been deposited electrochemically onto the surface of fluorine doped tin oxide (FTO) sheets for the analysis of malathion organophosphate (OP). The -COOH functionalization of MWCNTs has been done for the covalent immobilization of an enzyme acetylcholinesterase (AChE). The prepared PEDOT-MWCNTs/FTO and AChE/PEDOT-MWCNTs/FTO bioelectrodes were characterized by Fourier transform infrared spectrometry (FTIR), Field emission-scanning electron microscopy (FE-SEM) and electrochemical studies. Various optimization studies were done for different parameters including pH of 0.1 M phosphate buffer solution (PBS) (7.5), AChE concentration (50 mU), substrate concentration (0.3 mM) and inhibition time (10 min). The detection limit for malathion was calculated to be 1 fM within the linear range 1 fM to 1 mu M. The inhibited AChE could be regenerated to 99% by 2-PAM. The storage stability and reusability of the prepared bioelectrode is observed to be 30 days and seven times, respectively. Recoveries of malathion from the spiked lettuce sample ranged between 96-98% using the developed bioelectrode. (C) 2016 Elsevier B.V. All rights reserved.