Nile blue/graphene (NB-GNs) nanocomposite was synthesized for the first time via a green and effective one-step electrochemical method, allowing to reduce graphene oxide (GO) and NB on the glassy carbon electrode (GCE) simultaneously and construct GCE-GNs-NBpoly composite. The composite was characterized by scanning electron microscopy (SEM), UV-Vis spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The electrochemical results obtained in the absence of any redox probe, where NB was active, allowed to trace step-by-step addition of the NB-GNs nanocomposite onto the GCE electrode surface, supporting formation of the GCE-GNs-NBpoly composite. The electrocatalytic activity of the as-prepared GCE-GNs-NBpoly towards O-2 reduction was studied in neutral medium. The results revealed excellent electrocatalytic performance for two-electron reduction of oxygen, suggesting its potential application as metal-free electrocatalysts for O-2 reduction reaction. Application of the GCE-GNs-NBpoly in electrochemical biosensing was demonstrated by immobilization of glucose oxidase (GOx) on the surface of GCE-GNs-NBpoly, and then, using it for sensing of glucose. The biosensor exhibited a linear response, from 0.2 to 2.0 mM glucose, with a low detection limit, 2.1 mu M, and high sensitivity, 67.0 mu A mM (1)cm (2), obtained by cyclic voltammetry method. The proposed biosensor was successfully tested for determination of glucose in blood serum samples. (C) 2015 Elsevier Ltd. All rights reserved.