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　10(7)　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.