AbstractCarbon nanotubes (CNTs) have been used for various bioelectrochemical applications, presumably for substantial improvement in performance. However, often only moderate results observed, with many governing factors have been considered and suggested yet without much systematic evaluation and verification. In this study, CNT‐supported glucose oxidase (CNT–GOx) was examined in the presence of 1,4‐benzoquinone (BQ). The intrinsic Michaelis parameters of the reaction catalyzed by CNT–GOx were found very close to those of native GOx. However, the Nafion entrapment of CNT–GOx for an electrode resulted in a much lower activity due to the limited availability of the embedded enzyme. Interestingly, kinetic studies revealed that the biofuel cell employing such an enzyme electrode only generated a power density equivalent to <40% of the reaction capability of the enzyme on electrode. It appeared to us that factors such as electron and proton transfer resistances can be more overwhelming than the heterogeneous reaction kinetics in limiting the power generation of such biofuel cells. Biotechnol. Bioeng. 2009; 104: 1068–1074. © 2009 Wiley Periodicals, Inc.