Enzymatic biofuel cell (EBC) employing new catalyst for anode electrode is fabricated. The new catalyst consists of glucose oxidase (GOx), polyaniline (PANI) and carbon nanotube (CNT) that are multiply stacked together and finally the stack layer is surrounded by glutaraldehyde (GA) (GA/[GOx/PANI/CNT](n)). To evaluate how the GA/[GOx/PANI/CNT](n) layer affects EBC performance and stability, electrochemical characterizations are implemented. Regarding optimization, GA/[GOx/PANI/CNT](3) is determined. For elucidating reaction mechanism between glucose and flavin adenine dinucleotide (FAD) of GA/[GOx/PANI/CNT](3), associated investigations are performed. In the evaluations, drop in reduction current peak of FAD is observed with provisions of glucose and O-2, while glucose does not influence FAD reaction without O-2, confirming O-2 makes mediator role. When the GA/[GOx/PANI/CNT](3) layer is adopted, superior catalytic activity and EBC performance are gained (electron transfer rate constant of 5.1 s(-1), glucose sensitivity of 150 iA mM(-1) cm(-2), and EBC maximum power density (MPD) of 0.29 mW cm(-2)). Regarding EBC stability, MPD of EBC adopting GA/[GOx/PANI/CNT](3) maintains up to 93% of their initial value even after four weeks. Although GA is little effective for improving EBC performance, EBC stability is helped by GA due to its adhesion promotion capability with [GOx/PANI/CNT](n) layer. (C) 2015 Elsevier B.V. All rights reserved.