We present a simple and massively parallel nanofabrication technique to produce self-assembled periodic nanohole arrays over a millimeter-sized area of metallic film, with a tunable hole shape, diameter, and periodicity, Using this method, 30 x 30 mu m(2) defect-free areas of 300 nm diameter or smaller holes were obtained in silver; this area threshold is critical because it is larger than the visible wavelength propagation length of surface plasmon waves (similar to 27 mu m) in the silver film. Measured optical transmission spectra show highly homogeneous characteristics across the millimeter-size patterned area, and the), are in good agreement with FDTD simulations. The simulations also reveal intense electric fields concentrated near the air/silver interface, which was used for surface-enhanced Raman spectroscopy (SERS). Enhancement factors (EFs) measured with different hole shape and excitation wavelengths on the self-assembled nanohole arrays were 10(4)-10(6). With an additional Ag electroless plating step, the EF was further increased up to 3 x 10(6). The periodic nanohole arrays produced using this tunable self-assembly method show great promise as inexpensive SERS substrates as well as surface plasmon resonance biosensing platforms.