Enzymatic biofuel cell (EBC) employing a layer-by-layer (LbL) structure consisting of multiple layers of glucose oxidase (GOx) and poly(ethyleneimine) (PEI) at carbon-nanotube (CNT) ([GOx/PEl](n)/CNT) is fabricated. The [GOx/PEI](n)/CNT serves as anode catalyst for promoting glucose reaction, while Pt is employed as cathode catalyst. To evaluate effect of [GOx/PEl](n)/CNT on EBC performance and stability, several characterizations are conducted. The optimal GOx/PEI layer is determined electrochemically, and it turns out that [GOx/PEl](2)/CNT is the best. Electron transfer rate constant of the optimal layer is 11.3 s(-1), its glucose sensitivity is 83 mu AmM-1 cm(-2), and maximum power density of EBC adopting [GOx/PEI](2)/CNT is 1.34 mWcm(-2). The values are superior to those of other reference structures, indicating that the [GOx/PEI](2)/CNT can produce excellent reactivity, followed by improved EBC performance. In terms of redox reaction mechanism of flavin adenine dinucleotide (FAD) within [GOx/PEI](2)/CNT, glucose does not affect the redox reaction of FAD, while oxygen serves as mediator in transferring electrons and protons produced by glucose oxidation into those for reduction reaction of FAD. It is also found that the [GOx/PEI](2)/CNT is confined by surface reaction and the reaction is quasi-reversible. Regarding long-term stability, [GOx/PEl](2)/CNT maintains -83% of initial activity even after two weeks. (C) 2015 Elsevier B.V. All rights reserved.