Effects of various configurations of a chitosan film-templated biocathode on a direct electron transfer (DET)-type reaction were investigated. Self-standing electrospun carbon nanofibers (ECFs) obtained from the carbonization of polymer fibers were used as supporting material. The biocathode was built from a layered disposition of bilirubin oxidase (BOD) and multi-walled carbon nanotubes (MWCNTs) entrapped in chitosan matrix onto the surface of the ECFs. When the enzyme layer was sandwiched between two layers of MWCNTs, significant improvement of DET oxygen reduction occurred at pH 7.0, which was ascribed to the presence of local negative charges and to pi-pi interactions favoring enzyme orientation. The electrode preparation is simple and results in an efficient biocathode with BOD non-specifically attached to the porous material. The limiting current density of the optimized biocathode was 1.2 mA cm(-2). Combined to an abiotic Au60Pt40-modified anode, the resulting hybrid glucose biofuel cell delivered an open circuit voltage and a power density value of 0.9 V and 63 mu W cm(-2), respectively. (C) 2016 The Electrochemical Society.