Palladium nanoparticle-loaded carbon nanofibers (Pd/CNFs) were synthesized by the combination of electrospinning and thermal treatment processes. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images show that spherical Pd nanoparticles (NPs) are well-dispersed on the surfaces of CNFs or embedded in CNFs. X-ray diffraction (XRD) pattern indicates that cubic phase of Pd was formed during the reduction and carbonization processes, and the presence of Pd NPs promoted the graphitization of CNFs. This nanocomposite material exhibited high electric conductivity and accelerated the electron transfer, as verified by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The Pd/CNF-modified carbon paste electrode (Pd/CNF-CPE) demonstrated direct and mediatorless responses to H(2)O(2) and NADH at low potentials. The analytical performances of the Pd/CNF-CPEs towards reduction of H(2)O(2) and oxidation of NADH were evaluated. The high sensitivity, wider linear range, good reproducibility, and the minimal surface fouling make this Pd/CNF-CPE a promising candidate for amperometric H(2)O(2) or NADH sensor.