Fabricating well-defined and highly reproducible platforms for surface-enhanced Raman scattering (SERS) is very important in developing practical SERS sensors. We report a novel SERS platform composed of a single metallic nanowire (NW) on a metallic film. Optical excitation of this novel sandwich nanostructure provides a line of SERS hot spots (a SERS hot line) at the gap between the NW and the film. This single nanowire on a film (SNOF) architecture can be easily fabricated, and the position of hot spots can be conveniently located in situ by using an optical microscope during the SERS measurement. We show that high-quality SERS spectra from benzenethiol, brilliant cresyl blue, and single-stranded DNA can be obtained on a SNOF with reliable reproducibility, good time stability, and excellent sensitivity, and thus, SNOFs can potentially be employed as effective SERS sensors for label-free biomolecule detection. We also report detailed studies of polarization- and material-dependent SERS enhancement of the SNOF structure.