In this study, graphene nanofibers (GNF), which are a superior support material, are successfully synthesized via the dendritic unzipping of stacked-cup carbon nanofibers (SCNF). Ultrasmall Pd nanoparticles are uniformly dispersed on the GNF (Pd/GNF) via chemical reduction under mild conditions without any surfactant involved. The components and structure of Pd/GNF are evaluated via scanning electron microscopy (SEM), transmission electron microscopy (TEM), powder X-ray diffraction (XRD), Raman spectra and X-ray photoelectron spectroscopy (XPS). The characterization results indicate that the Pd nanoparticles have a uniform size of 3-6 nm without significant aggregation and the overall Pd content is about 11.2 wt% in the Pd/GNF composite. Moreover, a modified electrochemical sensor based on the Pd/GNF composite is successfully fabricated. In the two investigated redox probes (IrCl6 2- and [Fe(CN)6]3-), Pd/GNF shows a superior electrochemical response compared to the Pd nanoparticles loaded on SCNF and bare glass carbon electrode. For the detection of small biomolecules, Pd/GNF could individually or simultaneously detect ascorbic acid (AA), dopamine (DA) and uric acid (UA) through differential pulse voltammetry. The linear concentration ranges of UA, DA and AA are 0.1-1200 μM, 1-180 μM and 0.1-6000 μM, respectively.