A　novel　all-femtosecond-laser-processing　technique　is　proposed　for　the　fabrication　of　2D　periodic　metal　nanostructures　inside　3D　glass　microfluidic　channels,　which　have　applications　to　real-time　surface-enhanced　Raman　spectroscopy　(SERS).　In　the　present　study,　3D　glass　microfluidic　channels　are　fabricated　by　femtosecond-laser-assisted　wet　etching.　This　is　followed　by　the　space-selective　formation　of　Cu-Ag　layered　thin　films　inside　the　microfluidic　structure　via　femtosecond　laser　direct　writing　ablation　and　electroless　metal　plating.　The　Cu-Ag　films　are　subsequently　nanostructured　by　irradiation　with　linearly　polarized　beams　to　form　periodic　surface　structures.　This　work　demonstrates　that　a　double　exposure　to　laser　beams　having　orthogonal　polarization　directions　can　generate　arrays　of　layered　Cu-Ag　nanodots　with　dimensions　as　small　as　25%　of　the　laser　wavelength.　The　resulting　SERS　microchip　is　able　to　detect　Rhodamine　6G,　exhibiting　an　enhancement　factor　of　7.3　x　10(8)　in　conjunction　with　a　relative　standard　deviation　of　8.88%.　This　3D　microfluidic　chip　is　also　found　to　be　capable　of　the　real-time　SERS　detection　of　Cd2+　ions　at　concentrations　as　low　as　10　ppb　in　the　presence　of　crystal　violet.　This　technique　shows　significant　promise　for　the　fabrication　of　high　performance　microfluidic　SERS　platforms　for　the　real-time　sensing　of　toxic　substances　with　ultrahigh　sensitivity.