The flexible SERS substrate with high activity was prepared by electrospinning. The spinning solution was mixed with silver nitrate and polyvinyl alcohol in certain proportion. The nanofiber substrate was prepared by UV irradiation reduction method after spinning. The synthesized nanofiber substrate was characterized by Scanning electron microscopy (SEM), Transmission electron microscope (TEM), Fourier infrared spectroscopy (FT-IR), Raman spectroscopy, UV visible spectroscopy (UV-Vis). Research show that the silver nanoparticles are spherical in shape and less than 10 nm in diameter, distributed in composite fibers. Using rhodamine as probe molecule, the optimal SERS performance of the substrate is obtained, 16wt% of silver nitrate and 3 h of UV irradiation. At the same time, the substrate is applied to the detection of nicotinic acid drugs. The results show that the limit of Raman detection is 10-5 mol ? L-1.