To meet the needs of low-cost and easy-to-prepare surface enhanced Raman scattering (SERS) substrate in practical application, plasma technology was used to treat poly(methyl methacrylate) (PMMA) to form nanofiber structures on the surface, and combined with metal sputtering process, to prepare SERS substrate. On the basis of the theoretical analysis of the formation mechanism of PMMA nanofibers, rhodamine 6G (R6G) was used as the probe molecule to investigate the SERS performance of the substrate. The results show that the limit of detection of the SERS substrate is 10-10 mol/L, the enhancement factor is 1.75×106, and the relative standard deviation is 6.52%. Moreover, the SERS subsrtate can maintain good stability within 30 days, illustrating a great potential for practical application in the field of biochemical detection.