We have developed and characterized novel multilayered metal film-based surface-enhanced Raman scattering (SERS) substrates capable of enhancing SERS signals over an order of magnitude compared to conventional single layer substrates. In addition to enhanced signal intensity, these multilayered metal film substrates also exhibit longer SERS active lifetimes, higher reproducibility and lower detection limits than single layer silver substrates. Multilayered metal film substrates were fabricated by repeated vapor deposition of metal films over nanometer sized silica spheres. Different sizes of silica spheres were evaluated in order to obtain the optimal SERS enhancements. Meanwhile, different coating methods, drop coating and spin coating, were applied to form silica sphere layers that provided the roughness for SERS enhancements. These two coating methods were also compared for various silica sphere sizes by investigating their effects on the SERS enhancements. By applying additional silver layers on top of silver film over silica sphere SERS substrates, multi-layer enhancements can be observed. Additionally, different metals, such as gold, were used to further optimize the stability and reproducibility of these novel substrates. In order to speed up the fabrication of these multiple metal layer SERS substrates, silver oxide layers produced in an oven were investigated, reducing fabrication time by a factor of 50.© (2004) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.