Surface-enhanced Raman scattering (SERS) has shown high promise in analysis and bioanalysis, wherein noble metal nanoparticles (NMNPs) such as silver nanoparticles were employed as substrates because of their strong localized surface plasmon resonance (LSPR) properties. However, SERS-based pH sensing was restricted because of the aggregation of NMNPs in acidic medium or biosamples with high ionic strength. Herein, by using the electrostatic interaction as a driving force, AgNPs are assembled on the surface of ethylene imine polymer (PEI)/poly(vinyl alcohol) (PVA) electrospun nanofibers, which are then applied as highly sensitive and reproducible SERS substrate with an enhancement factor (EF) of 10(7)-10(8). When p-aminothiophenol (p-ATP) is used as an indicator with its b(2) mode, a good and wide linear response to pH ranging from 2.56 to 11.20 could be available, and the as-prepared nanocomposite fibers then could be fabricated as excellent pH sensors in complicated biological samples such as urine, considering that the pH of urine could reflect the acid base status of a person. This work not only emerges a cost-effective, direct, and convenient approach to homogeneously decorate AgNPs on the surface of polymer nanofibers but also supplies a route for preparing other noble metal nanofibrous sensing membranes.