In this paper, we propose a facile and cost-effective electrospinning technique to fabricate surface-enhanced Raman scattering (SERS) substrates, which is appropriate for multiple analytes detection. First of all, HAuCl∙3HO was added into the TEOS/PVP precursor solution, and flexible SiO nanofibers incorporated with gold nanoparticles (SiO@Au) were prepared by electrospinning and calcination. Subsequently, the nanofibrous membranes were immersed in the tannic acid and 3-aminopropyltriethoxysilane solution for surface modification through Michael addition reaction. Finally, the composite nanofibers (Ag@T-A@SiO@Au) were obtained by the in-situ growth of Ag nanoparticles on the surfaces of nanofibers with tannic acid as a reducing agent. Due to the synergistic enhancement of Au and Ag nanoparticles, the flexible and self-supporting composite nanofibrous membranes have excellent SERS properties. Serving as SERS substrates, they are extremely sensitive to the detection of 4-mercaptophenol and 4-mercaptobenzoic acid, with an enhancement factor of 10. Moreover, they could be utilized to detect analytes such as pesticide thiram at a low concentration of 10 mol/L, and the substrates retain excellent Raman signals stability during the durability test of 60 days. Furthermore, the as-fabricated substrates, as a versatile SERS platform, could be used to detect bacteria of without a specific and complicated bacteria-aptamer conjugation procedure, and the detection limit is up to 10 colony forming units/mL. Meanwhile, the substrates also show an excellent repeatability of SERS response for organelles. Briefly, the prime novelty of this work is the fabrication of Au/Ag bimetallic synergetic enhancement substrates as SERS platform for versatile detection with high sensitivity and stability.