Heavy metal pollution in developing countries urgently becomes a serious environmental issue due to rapid industrial development. Therefore, to detect the trace of heavy metal ions in water and food is very critical for environmental governance and human health. Surface-enhanced Raman scattering (SERS) based on electromagnetic field extraordinary enhanced in the proximity of metal nanostructures can generate strong Raman scatting, which is an effective method for trace detection. Most of SERS devices have been fabricated on the solid substrate surfaces, while detection of toxic substances in the close environment is much more preferable. In this paper, we propose the novel technique that can create 2D periodic metal (Cu-Ag) nanostructure inside 3D glass microfluidic channel by all-femtosecond-laser-processing. Specifically, after fabrication of 3D glass microfluidic channel by femtosecond laser assisted wet etching, Cu-Ag thin films are formed inside the microfluidic channel by femtosecond laser selective metallization. The thin films are then 2D nanostructured by femtosecond laser induced periodic surface structure (fs-LIPSS) formation. The dimension of nanostructured Cu-Ag film is quarter of laser wavelength. By testing with rhodamine 6G, we demonstrate the fabricated microchips can be used as a sensitive SERS device with an enhancement factor larger than 107 and 8.8% relative standard deviation. Consequently, the Real-time SERS detection in microfluidic chips was successfully demonstrated, which verified capability of the fabricated microchips as an excellent microfluidic SERS platform with ultrasensitive and uniform enhancement.