Carbon nanofibers (CNFs) are a promising material as conducting support of catalysts in (bio)electrochemical applications. Self-standing CNFs result from the carbonization at 1200 °C of electrospun polymer fibers with mean fiber diameter of 330 ± 50 nm. Such felts present interesting properties like fibrous and porous morphology to relieve the mass-transfer limitation of substrates and to provide high loadings of catalysts to enhance the electrochemical performances of the resulting electrodes. We show the beneficial feature of the CNFs as support compared with carbon dense structure for efficient immobilization of either abiotic catalysts based on metal nanoparticles or enzyme as biological catalyst. More specifically, palladium or platinum modified gold nanocatalysts remarkably boost the glucose electro-oxidation when deposited onto CNFs. Similarly, the immobilization of the bilirubin oxidase enzyme on the porous CNFs induces significant improvement of the mediated oxygen electroenzymatic reduction. The advances presented in this work show the high performance of the electrospun carbon fiber electrodes as promising materials for abiotic and enzymatic catalysis for the development of hybrid biofuel cells.