Co-polymer of pyrrole and ferrocenecarboxylate modified pyrrole (P(Py-FcPy)) has been prepared by electrochemical polymerization on indium-tin-oxide (ITO) coated glass surface and glucose oxidase (GOx) as model enzyme has been entrapped during deposition for fabrication of mediator-less electrochemical biosensor. The 4-(1H-pyrrol-1-yl)phenyl ferrocenecarboxylate used for copolymerization has been synthesized chemically and characterized using H-1 NMR and C-13 NMR techniques. Presence of ferrocene moiety ensure the redox activity in P(Py-FcPy) for faster electron shuttling. The GOx-P(Py-FcPy)/ITO electrodes thus fabricated has been characterized using cyclic voltammetry (CV) and used for glucose concentration estimation in standard solutions using CV and amperometric techniques. Bio-electrode exhibit linearity up to 16.8 mM, lower detection limit of 0.17 mM, Michaelis-Menten constant, K-M(APP), of 1.6 mM and sensitivity of 19.21 mu A mM(-1). The results indicate the feasibility of fabricating sensitive mediator-less electrochemical biosensors using ferrocene modified polypyrrole film. (C) 2011 Elsevier B.V. All rights reserved.