In this work, we report a quick and controllable method for fabricating Ag nanoparticles (NPs) and nano-assemblies (NAs) on silicon (Si) surface via a galvanic reaction assisted by surfactant hexadecyltrimethylammonium bromide (CTAB). Obtained Si substrates with high density Ag NAs serve as surface-enhanced Raman scattering (SERS) substrates for unlabelled molecular sensing. The addition of CTAB enables not only the electromagnetic enhancement by controllable producing Ag NAs “hotspots”, but also the signal enhancement by enriching probe molecules via hydrophobic interaction. Two concentration regimes of 1.0 × 10−8–1.0 × 10−11 M and 1.0 × 10−12–1.0 × 10−16 M have been obtained for quantitative and qualitative detection of Rhodamine 6G (R6G) in aqueous solution, respectively. As a result, for determination of R6G, the quantitative and qualitative detection limits of 1.0 × 10−11 and 1.0 × 10−16 M have been obtained, respectively, with high reproducibility showing the relative standard deviation (RSD) of ∼7.3%. Simulation has been carried out using finite-difference time-domain (FDTD) to evaluate the dependence of electric field on the size and inter-gap of Ag NPs, showing good agreement with experimental results. The high sensitivity also enables SERS characterization of non-resonant and low Raman cross-section biomolecules, for example, amino acids. Such a fabrication process is simple, fast, cheap and reproducible; and therefore, it would be helpful to produce high sensitive SERS substrates to broaden their applications in chemical and biological fields for molecular diagnostics to single-/multiple-molecule detection.