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, FT-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 × 10− 4 mA mg− 1 dL and 3.887 × 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.