In this study, a novel approach for the fabrication of a biosensor utilizing a conducting polymer and silver nanowires is reported. To obtain immobilization platform for butyrylcholinesterase (BChE), a graphite electrode was modified with the poly(5,6-bis(octyloxy)-4,7-di(thieno[3,2-b]thiophen-2yl)benzo[c][1,2,5]oxoadiazole) (PTTBO) which has a hydrophobic alkyl chain as the pendant group providing hydrophobic nature to the matrix. Since biomolecules contain both hydrophobic and hydrophilic parts in their structure, alkyl chains interact with the proteins which provide an enhanced stability. Biosensor performance was improved through the deposition of silver nanowires on the polymer coated surfaces which enhances the charge transfer rate. This enabled the development of rapid, highly sensitive and stable amperometric sensors for the quantitative determination of organophosphorus pesticide; paraoxon. Fabricated biosensor showed two linear ranges between 0.5-8 mu M and 10-120 mu M with a low detection limit of 0.212 mu M when butyryl thiocholine iodide is used as the substrate. Surface modifications were monitored by scanning electron microscope (SEM) and cyclic voltammetry (CV) techniques. Under optimal operational parameters, fabricated sensors were tested for paraoxon detection in milk and tap water based on the inhibition of the enzyme molecules, where recovery tests proved the applicability of the designed system. (C) 2016 Elsevier B.V. All rights reserved.