Glucose oxidase (GOD) and cytochrome c (Cyt c) were co-entrapped in the poly(diallyldimethylammonium chloride)-graphene nanosheets-gold nanoparticles (PDDA-Gp-AuNPs) hybrid nanocomposites modified glassy carbon electrode to prepare a novel bi-protein bio-interphase. Electron transfer and electrocatalysis of the bi-protein bio-interphase were investigated in detail. The results showed that the PDDA-Gp-AuNPs nanocomposites accelerated the electron transfer between proteins and electrode. The bi-protein exhibited effective direct electron transfer (DET) reaction with an apparent rate constant (k(s)) of 2.36 s(-1). The optimal molar ratio and total amount of Cyt c and GOD in the bio-interphase for DET of GOD was estimated to be about 3:1 and 1.40 nmol, respectively. The bi-protein bio-interphase could be used to detect glucose based on the consumption of 02 with the oxidation of glucose catalyzed by GOD. The resulted biosensor exhibits wide linear range from 2.0 to 18.0 mM. Thus, this study not only successfully achieved DET of GOD, but also constructed a novel biosensor for glucose detection. (C) 2013 Elsevier Ltd. All rights reserved.