Electrophoretically deposited nano-structured polyaniline (NS-PANI) film has been utilized for fabrication of glucose biosensor by covalent immobilization of glucose oxidase (GOx) using N-ethyl-N-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide chemistry. This GOx/NS-PANI/ITO bioelectrode has been characterized using scanning electron microscopy, Fr-IR, UV-Visible spectroscopy and differential pulse voltammetry (DPV) techniques. The response studies carried out on GOx/NS-PANI/ITO bioelectrode using DPV and photometric studies reveal linearity up to 400 mgdL(-1) with sensitivity as 1.05 x 10(-4) mA mg(-1) dL and 3.887 x 10(-5) Abs mg(-1) dL, respectively. The lower value of Michaelis-Menten constant obtained for immobilized GOx (2.1 mM) compared with that of free GOx (5.85 mM) suggests high affinity of enzyme to this matrix. (C) 2010 Elsevier B.V. All rights reserved.