The surface of polytetrafluoroethylene (PTFE) was ablated by femtosecond laser, and then the surface enhanced Raman scattering (SERS) substrate was prepared by depositing silver nanoparticles on the surface of laser structured PTFE substrate. By adjusting the scanning path of femtosecond laser, PTFE substrates with one-dimensional grating structure and two-dimensional grating structure are obtained, respectively. After coating a thin layer of silver nanoparticles, the two-dimensional grating structure of polytetrafluoroethylene substrate can serve as a SERS substrate, which shows much better SERS enhancement, about three times higher than that based on one-dimensional grating structure.