Despite extensive advances in plasmonic architectures, the development of simple, reproducible, and cost-effective surface-enhanced Raman scattering (SERS) platforms for high-performance molecular sensing remains a significant challenge. Herein, we developed surface-roughened Ag wires with a characteristic of uniformly distributed plasmonic elements of size 30 to 100 nm for enabling highly sensitive SERS applications. The closely spaced nanostructures on the Ag wire surface significantly enhanced the light-matter interaction above a wavelength of 550 nm, indicating the formation of plasmonic hotspots due to the interparticle plasmon coupling. They were successfully utilized as SERS-based sensor platforms to attain a low detection level of 1 nM with an enhancement factor, EF up to 108 for methylene blue molecules. The versatility of these SERS sensors was confirmed using different analyte molecules, including sulfur-containing pesticide Thiram, methyl orange, and rhodamine 6G. In addition, these SERS substrates exhibited excellent signal uniformity, with relative standard deviations (RSDs) of Raman intensities below 15%, indicating their high reproducibility and reliability. The numerical optical simulations based on the finite-difference time-domain (FDTD) method confirmed the polarization-independent light localization capability in surface-roughened Ag wires with the E-field intensity enhancement factor, |E|2/|E0|2, to exceed 103, revealing their outstanding plasmonic activity. Furthermore, the density functional theory (DFT) calculations on the Ag/analyte interface revealed superior adsorption characteristics with an adsorption energy, Eads, of −0.533 Ry. Therefore, the enhanced performance of the proposed SERS substrate emphasizes its strong potential for enabling low-cost sensor technologies capable of highly sensitive, label-free molecular detection and identification.