A novel amperometric biosensor based on polypyrrole (PPy) nanotube array deposited on a Pt plated nano-porous alumina substrate and its performances are described. Glucose oxidase (GOx) enzyme was selected as the model enzyme in this study. Commercially available nano-porous alumina discs were used to fabricate electrodes in order to study the feasibility of enzyme entrapment by physical adsorption. A PPy/PF6− film comprising of nanotube array was synthesized using a solution containing 0.05M Pyrrole and 0.1M NaPF6 at a current density of 0.3 mA/cm2 for 90 s. The immobilization was done by physical adsorption of 5 µL of GOx (from a stock solution of 2 mg/mL of 210 U/mg) on each electrode. A sensitivity of 7.4mAcm¯²M¯¹ was observed with PPy nanotube array where the maximum tube diameter was 100 nm. A linear range of 500µM–13mM and a response time of about 3 s were observed with a nanotube array where the maximum tube diameter was 200 nm. The synthesized nanotube arrays were characterized by galvanostatic electrochemical technique. Calculated value of apparent Michaelis–Menten constant (Km) was 7.01 mM. The use of nano-porous template electrodes leads to an efficient enzyme loading and provides an increased surface area for sensing the reaction. These factors contribute to increase the characteristic performances of the novel biosensor.