In this study, by utilizing a two-step route of electrospinning and polyol immersion, in the absence of any surfactant or sensitizing and stabilizing reagent, a well-distributed assembly of Ag NPs on the electrospun polyurethane (PU) nanofibers was successfully fabricated through a simple and controllable manner. Based on the FE-SEM, XRD and FT-IR analyses, the polyol medium plays an important role in the growth of highly monodispersed Ag NPs, wherein the hydroxyl group of ethylene glycol (EG) can be bridged to the amide group on the surface of the PU nanofibers through intermolecular hydrogen bonds. Fabrication of a polymer fibrous membrane effectively attached/decorated with noble metal NPs, which is essential as flexible and high efficiency substrates for SERS application where the molecule analytes are directly adsorbed on their surfaces is important, could be realized by the present electrospun PU-Ag(EG) nanofibers, employing 4-mercaptobenzoic acid (4-MBA) as probe molecules.