A facile strategy for the fabrication of electrochemical biosensors by immobilizing well-dispersed palladium nanoparticles (PdNPs) on the water-stable polyvinyl alcohol/polyethyleneimine (PVA/PEI) nanofibers through the combination of electrospinning technique and the process of in-situ reduction has been demonstrated. The synthesized PdNPs/(PVA/PEI) nanocomposites were characterized by field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The experimental results confirmed that the PdNPs immobilized on the electrospun PVA/PEI nanofibers with an average diameter of 3.4 nm were well-dispersed, which could be ascribed to the complexation between Pd(II) and the free amine groups of PEI. Further investigations suggested that the PdNPs/(PVA/PEI) nanocomposites with well-separated PdNPs and large surface area exhibited high performance as electrochemical biosensors to detect hydrogen peroxide (H2O2).