To develop a smart free-standing surface enhanced Raman scattering (SERS) substrate, silver nanoparticles (AgNPs) embedded temperature-sensitive nanofibrous membrane was fabricated by electrospinning their aqueous solution containing the copolymer poly(N-isopropylacrylamide-co-Nhydroxymethylacrylamide), followed by heat treatment to form crosslinking structure within its constituent nanofibers. To avoid negative effect of the additive like stabilizer and the reactant like reductant on their SERS efficiency, the AgNPs were in-situ synthesized through reducing Ag+ ions dissolved in the polymer solution by ultraviolet irradiation. The prepared hybrid nanofibrous membrane with high stability in aqueous medium can reach its swelling or deswelling equilibrium state within 15 seconds with the medium temperature changing between 25 ℃ and 50 ℃ altately. When it was used as a free-standing SERS substrate,10-12 mol/L of 4-nitrothiophenol in aqueous solution can be detected at room temperature, and elevating detection temperature can further lower its low detection limit. Since its generated SERS signal has desirable reproducibility, it can be used as SERS substrate for quantitative analysis. Moreover, the hybrid membrane as SERS substrate is capable of real-time monitoring the reduction of 4-nitrothiophenol into 4-aminothiophenol catalyzed by its embedded AgNPs, and the detected intermediate indicates that the reaction proceeds via a condensation route.