This work presents a 3D carbon composite electrode matrix, consisting of reduced graphene oxide (RGO), carbon nanotubes, and carbon fiber felt. In this matrix, the edges of the RGO sheets are bounded to cup-stacked carbon nanotubes (CSCNTs) while the micrometer scale basal planes of the RGO sheets are suspended between the fibers. The presence of free-standing RGO sheets in which it orbitals of the basal planes do not interact with the electrode surface offers advantageous conditions to explore demanding bioelectrochemical reactions, such as the FADH(2)/FAD redox transition often claimed in the literature as originated from a direct electron communication between micro/nano carbon based electrodes and glucose oxidase (GOx). At least, three important questions rise from this system: (i) how the junction between the carbon nanostructures and the carbon microfibers affects the monitored electron transfer? (ii) are the monitored Faradaic peaks originated from native or denaturated GOx? (iii) is GOx still active for glucose sensing after suffering conformational changes due to the adsorption process on the surface of the electrode? It is intended here to contribute with the investigation of these questions. According to our results, the electronic interactions between CSCNTs and RGO sheets seem to modulate the performance of the aforementioned reaction, as improved kinetic parameters were observed on the 3D composite matrix rather than the obtained with 3D electrodes modified with CSCNTs or RGO sheets themselves. However, spectroscopic experiments suggested that GOx suffers significant conformational changes when adsorbed on the surface of the carbon nanostructures, leading to the denaturation of the protein. Even though the electrochemical experiments indicate that the denaturation of the enzyme does not seem to completely release the FAD moiety, the operational capability of the resulting glucose biosensor was greatly compromised. (C) 2016 Elsevier B.V. All rights reserved.