A new electrochemical biosensor based on the covalent immobilization of two enzymes on self-assembled monolayer attached to a polycrystalline gold electrode is proposed, experimentally. The acetylcholinesterase (AChE) and cholineoxidase (ChO) are two enzymes that covalently co-immobilized on the mercaptopropionic acid self-assembled monolayer on polycrystalline gold electrode (Au-MPA-AChE/ChO SAM). Fabrication steps and electrochemical interaction of the Au-MPA-AChE/ChO SAM with carbaryl were monitored by general electrochemical methods like cyclic voltammetry (CV) and chronoamperometry (CA), and by a more advanced method, electrochemical impedance spectroscopy (EIS) in the presence of parabenzoquinone (PBQ) or K-3[Fe(CN)(6)] redox probes. The close distances between two immobilized enzymes on Au-MPA SAM result in preconcentration of choline, the product of acetylcholine hydrolysis by AChE, in ChO nano-environment. Enzyme activity was measured by chronoamperometric tracing of hydroquinone from the reduction of PBQ mediator which in turn is oxidized at Au electrode in diffusion layer. Carbaryl was chosen as a model toxin and its inhibition characteristics were utilized for the toxin detection. The linear range for the determination of Carbaryl was 10-1000 nmol l(-1). A limit of detection 5.96 nmol l(-1) was obtained. (C) 2013 Elsevier Ltd. All rights reserved.