Methylene blue (MB) exhibited the stabilization effect toward an adsorbed glucose oxidase (GOx) on carbon-felt (CF), which is a microelectrode ensemble of micro carbon fibers (ca. 7 mu m in diameter) possessing a random three-dimensional structure. The bioelectrocatalytic activity was evaluated by cyclic voltammetry under a deoxygenated atmosphere using hydroquinone (HQ) as an electron transfer mediator. When GOx was adsorbed from the GOx-dissolved solutions [e.g., pure water, 1, 10, and 100 mM phosphate buffers (pH 6.8), 100 mM NaCl and KCl solutions], the adsorbed GOx showed almost no apparent bioelectrocatalytic activity, probably due to mainly adsorption-induced unfavorable conformational change and/or unfavorable enzyme orientation on the surface with the active site unavailable for substrate and mediator. In contrast, when the GOx was adsorbed from the mixed solutions of GOx and MB, the adsorbed GOx showed sufficient HQ-mediated bioelectrocatalytic activity. As adsorption solution, lower ionic strength solutions (e.g., pure water and 1 mM phosphate buffer, pH 6.8) seemed to be preferable to obtain larger activity. Simultaneous adsorption from the mixed solution of GOx and MB was much effective than the step-by-step adsorption. Binding interaction of MB with GOx in aqueous solution was confirmed by UV-vis and circular dichroism spectroscopy. (C) 2012 The Electrochemical Society. [DOI: 10.1149/2.jes036205] All rights reserved.