Electrospun carbon nanofibers (CNFs) were prepared by electrospinning of a polyacrylonitrile solution followed by convenient stabilization and carbonization thermal treatments. By observations of scanning electron microscopy, a mat of interlaced continuous submicronic fibers was detected. Characterization by Raman spectroscopy demonstrated the successful preparation of a carbon material and the influence of the heating-treatment temperature on the structure of the resulting CNFs. The novelty of the approach relies in the easy handling of the resulting electrospun mat electrode that does not require addition of any polymer binder or conductive material. This is the first time that electrospun conductive fibers are envisaged as electrode supports for redox enzymes immobilization applied to bioelectrocatalytic O₂reduction. The most interesting thing is the remarkable benefit effect of the CNFs on the electrical performances of the electrode which can ascribe to high loading of active enzymes and fast kinetics at the electrode surface. The remarkably intensified current density (∼1 mA cm⁻²) achieved at the enzymatic CNFs bioelectrode is probably due to the unique nanostructure and surface properties of these nanomaterials that make them promising candidates as enzymatic cathodes in biofuel cell devices.