Glucose oxidase (GOx) was immobilized onto glassy carbon electrode (GCE) that modified by reduced graphene oxide-gold nanoparticles- poly neutral red (RGO/AuNPs/PNR) nanocomposite. The composite was analyzed by scanning electron microscope (SEM), energy dispersive x-ray (EDX) spectroscopy, atomic force microscopy (AFM), attenuated total reflectance (ATR), cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS). SEM/EDX analysis showed the morphological of the nanocomposite. AFM results showed the morphology and structure of the RGO/AuNPs and RGO surfaces. The covalent bonding between glucose oxidase and composite was confirmed by ATR technique. The electrochemical experiments were done in 100 mM phosphate buffer at pH 7 and temperature of 25 degrees C with three electrodes including Ag/AgCl, platinum wire and the modified GCE as the reference electrode, the auxiliary electrode and working electrode respectively. The electrochemical results confirmed the activity and direct electron transfer of immobilized enzyme. The immobilized electroactive GOx concentration was estimated 3.06 x 10(-11) mol cm(-2). The results showed the immobilized enzyme had a good stability and maintained 90% of its performance after two weeks. The nano composite bioanode in an air-birthing biofuel cell and 100 mM glucose concentration showed 176 mu Wcm(-2) Power density. This strategy could be used for GOx-based biofuel cells. (C) 2017 Elsevier Ltd. All rights reserved.