We describe here the covalent modification of glassy carbon electrode with nile blue (NB), a redox dye, by electrochemical reduction of its corresponding diazonium salt, which is generated in situ from the reaction between the aromatic amino phenyl group of NB and sodium nitrite. NB is attached directly to the electrode surface without any cross linking agent or complex matrices. This modification method is very rapid, simple, in one step and provides a very stable grafted NB film. Cyclic voltammetry and electrochemical impedance spectroscopy were used to trace the reaction. The resulting NB films have a very stable and reversible electrochemical response and exhibit excellent electrocatalytic behavior toward hydrogen peroxide and oxygen reduction. The amperometric detection of hydrogen peroxide is performed at -0.15 V vs. Ag/AgCl. This strong catalytic effect for reduction of hydrogen peroxide and oxygen exhibited a biocompatible platform for development of glucose biosensors. Therefore, a glucose biosensor is developed using glucose oxidase by simple casting method based on decreasing of cathodic peak current of oxygen. This biosensor has been successfully applied to determination of glucose in human plasma sample. The great stability and reusability, excellent electrochemical reversibility, technically simple and possibility of preparation at short period of time make this method suitable for low-cost bioelectronical devices. (C) 2013 Elsevier B.V. All rights reserved.