AChE based biosensor for the determination of methyl parathion and monocrotophos directly in the standard solutions and spiked (water and fruit) samples have been evaluated and electrochemically characterized by cyclic voltammetric technique. The biosensor was applied for the direct determination of pesticides in spiked samples at ppb concentration levels without any sample pretreatment steps. The fabricated acetyl cholinesterase based biosensor entrapped using silica sol-gel onto the carbon paste electrode was used as a working electrode. Acetylcholinesterase catalyses the cleavage of acetylthiocholine chloride (ASChCl or substrate) to thiocholine and it was electrochemically oxidized giving significant anodic peak current around at 0.60 V versus saturated calomel electrode. The effect of substrate concentration, enzyme loading and pH on the response of the biosensor was investigated. The electrochemical experiments were performed in 0.1 M phosphate buffer solution (pH 7.0) and 0.1 M KCl at room temperature. The limit of detection and limit of quantification values were found to be 0.04 ppb, 47 ppb and 0.14 ppb, 158 ppb for methyl parathion and monocrotophos, respectively. The analytical curve obtained for methyl parathion and monocrotophos in pure solutions showed excellent linearity in the concentration range of 0.1-1.0 ppb and 50-950 ppb with an incubation time of 10 min and 4 min. The constructed sol-gel biosensor requires less time for analysis and no preconcentration/extraction was needed. (C) 2011 Elsevier B.V. All rights reserved.