The excellent catalytic activity of a novel carbon nanomaterial, soluble carbon nanofiber (CNF) with good dispersion and wettability, to the oxidation of dihydronicotinamide adenine dinucleotide (NADH) was described for biosensing application. The edge plane sites and oxygen-rich groups presented on the CNF surface could be partially responsible for its electrocatalytic behavior, which induced a substantial decrease by 573 mV in the overpotential of NADH oxidation reaction (compared to a bare electrode). The CNF-coated electrode thus allowed highly sensitive amperometric detection of NADH with a low limit of detection (0.11 muM), low applied potential (+0.06 V), and minimization of surface fouling. Such ability of CNF to promote the electron transfer between NADH and the electrode suggested a new, promising biocompatible platform for development of dehydrogenase-based amperometric biosensors. With alcohol dehydrogenase (ADH) as a model, the ADH/CNF-modified electrode could be constructed by a simple casting process. The proposed biosensor showed rapid and highly sensitive amperometric response to ethanol with acceptable preparation reproducibility and excellent stability.