Surface-enhanced Raman scattering (SERS) has powerful capabilities in the field of environmental analysis and biological diagnostics because of its instinctive properties of high sensitivity and label-free detection. However, the fabrication of SERS substrate requires complicated processes and expensive equipment. This paper proposes a simple method approach to synthesize a 3D SERS substrate via electroless galvanic replacement reaction inside a microfluidic channel. Copper microparticles are assembled in a microfluidic channel, and silver nitrate solution is pumped into the channel to in situ produce the silver nanoparticles (Ag NPs) on the surface of copper microparticles. Because the copper particles occupy the channel by stack, the 3D Cu@AgNP SERS substrate can be obtained. The probing molecule (methylene blue) was utilized to investigate with the limit of detection (1×10-7 M). The biological molecules (urea and melamine) have been used to demonstrate its benefits in medical applications, and cancer cell detection has been implemented to demonstrate its benefits in cell biology. In addition, the device can filter and wash cells, forming a simple and fast filter. Our work on this simple fabrication method of active SERS substrate has great value for medical and biological applications.