A carbon nanotubes/Ag nanoparticles (CNTs/AgNPs) surface-enhanced Raman scattering (SERS)-active substrate is prepared by the chemical reduction method, and a polydimethylsiloxane (PDMS) microfluidic system exhibiting SERS activity is constructed by using the molding method. Further, experiments are conducted by using the rhodamine 6G (R6G) solution at a concentration of 10 ^-7 mol/L as probe molecules. The experimental results verify the feasibility of the Raman test of the CNTs/AgNPs-enhanced substrate in the microchannel; in addition, the variation of the Raman signal of the probe molecules in the microchannel has also been verified and analyzed at different positions and time.