A surface-enhanced Raman scattering (SERS)-active microfluidic device with tunable surface plasmon resonances is presented here. It is constructed by silver grating substrates prepared by two-beam laser interference of photoresists and subsequent metal evaporation coating, as well as PDMS microchannel derived from soft lithography. By varying the period of gratings from 200 to 550 nm, surface plasmon resonances (SPRs) from the metal gratings could be tuned in a certain range. When the SPRs match with the Raman excitation line, the highest enhancement factor of 2×107 is achieved in the SERS detection. The SERS-active microchannel with tunable SPRs exhibits both high enhancement factor and reproducibility of SERS signals, and thus holds great promise for applications of on-chip SERS detection.