Reducing the physical adsorption of enzymes on carbon black and their resultant deactivation are the key factors to increasing the current density of enzymatic biofuel cells, and thus enabling their use in a wide range of applications. The effect of the surface treatment of carbon black on the physical adsorption of glucose oxidase (GOx) was investigated. GOx was then immobilized on a high-surface-area three-dimensional carbon electrode using a method other than physical adsorption. Acid treatment followed by graft polymerization of an anionic polymer and the mixing of carbon black with polyethylene glycol ( PEG) were both effective in reducing the physical adsorption of GOx. The combination of the surface modification using PEG and the immobilization of GOx via ammonium sulfate precipitation with crosslinking increased the glucose-oxidation current density obtained in an electrode fabricated using poly(vinylferrocene-co-acrylamide)-grafted carbon black. (C) 2014 The Electrochemical Society. All rights reserved.