Surface-enhanced Raman scattering (SERS) is a non-invasive technique that enables the detection and characterization of both small organic and big biological molecules at very low concentrations. To overcome the reproducibility issue of stochastically aggregated colloidal nanoparticles in SERS, various periodic metallic nanostructures created by self-assembly have been exploited. Unfortunately, most of these bottom-up approaches are limited in creating centimeter-sized samples, and are not compatible with standard microfabrication, thereby impeding the cost-efficiency and scale-up of these unconventional methodologies. Here we report a simple yet scalable templating technique for fabricating wafer-scale SERS substrates with high (> 108) and reproducible enhancement factors. Non-closed-packed colloidal crystals prepared by a spin-coating technique are used as templates to create periodic arrays of nano-dimples, nano-pyramids, and nano-flasks. These nanostructures have high-density sharp features (circular edges or nanoscale tips) that function as “hot spots” to concentrate electromagnetic field. We have also developed a finite-element method (FEM) model to simulate the spatial distribution of the electromagnetic field and predict the corresponding SERS enhancement factor from the periodic metallic nanostructures. Electrochemical SERS detection has also been investigated to concentrate the charged analytes (e.g., various biomarkers and dye molecules) in the vicinity of the sharp edges or tips by electrophoresis to further increase the sensitivity of the detection.