Hybrid femtosecond laser processing, which consists of femtosecond laser assisted wet etching, selective metallization, and laser induced periodic surface structure (LIPSS) formation, has enabled fabricating three-dimensional microfluidic surface enhanced Raman scattering (SERS) chips for highly sensitive sensing of a tiny amount of substances. To investigate dependence of laser wavelength on the period of ripple which strongly affects the enhancement factor for SERS, two different wavelengths (515 nm and 1030 nm) of femtosecond laser beams have been employed. The metal layers were formed in a closed glass microchannel by selective metallization technique composed of femtosecond laser direct ablation and successive electroless plating. Femtosecond laser direct writing was able to control the morphology of nanoripple on the metal layers by laser parameters, resulting in formation of homogenous high spatial frequency LIPSS (HFSL). The SERS results presented that the nanoripple with narrower groove fabricated by 515 nm femtosecond laser more enhanced the SERS intensity and the SERS analytical enhancement factor was evaluated to exceed 1 × 10⁸. Furthermore, we developed a novel method termed liquid-interface assisted surface enhanced Raman scattering (LI-SERS) to realize extremely sensitive sensing, which achieved detection limit of aM of R6G. The LI-SERS was able to locally aggregate the analyte molecules by laser irradiation at the interface between air and analyte solution containing the analytes in microchannel. The aggregation of analytes forced the molecules to enter into the “hot-spots” by Marangoni effect, which extraordinarily increased the SERS intensity. Amazingly, LI-SERS realized extremely sensitive sensing with analytical enhancement factor exceeding 1 × 10¹³.