In the present study polyvinyl alcohol (PVA) capped AgNPs (AgNPs@PVA) were used as nanocarrier for fabrication of an acetylcholinesterase (AChE) biosensor for organophosphate (OP) pesticide detection. AChE enzyme was immobilized via glutaraldehyde on drop casted AgNPs@PVA layer on glassy carbon electrode (GCE) for fabrication of the electrochemical biosensor. Through differential pulse voltammetry (DPV) the biosensor measured peak current at 0.6 V generated by AChE mediated hydrolysis of acetylthiocholine (ATCl) to thiocholine and its subsequent oxidation to dimer. By hindering AChE hydrolysis activity of the sensor probe by a model OP pesticide, malathion, the pesticide biosensor was developed as the inhibition effect was found to be proportional to the concentration of malathion. The fabricated biosensor exhibits linearity at range of 0.01 to 1 ng/mL of malathion at optimal conditions with detection limit (LOD) of 2.6 pg/mL. The fabricated AChE biosensor showed good selectivity for OP pesticides and exhibited good efficiency in real sample analysis.