Surface-enhanced Raman scattering (SERS) with enormous enhancements has shown great potential in single-molecule detection, however, the fabrication of large-scale, controllable and reproducible substrates with high SERS activity is a major limitation. This paper provides method to create wafer level SERS substrate with tunable nanoparticle sizes and interparticle gaps. Silver films with different thicknesses were deposited by electron beam evaporation (EBE) and annealed at 260 °C for 15min in nitrogen gas. When annealed, the thin Ag films break up under surface tension to form isolated nanoparticle. The nanoparticle size, density, and shape are found to be dependent on the thickness of Ag and the condition of annealing. The selective deposition of Ag nanoparticle on the silicon substrate is applied to create SERS active sites before the integration with a PDMS microfluidic chip which functions as a sample delivery device and a transparent optical window for SERS. Detections of Rhodamine 6G SERS spectra are accomplished by using a 633nm laser with 300W excitation power. The results show that the fabrication protocol of such a SERS substrate is low-cost, easy-fabrication and inexpensive. Therefore, this substrate may anticipate a wide range of applications in SERS-based sensors.