The analytical and physical properties are reported for nanohole arrays prepared with glancing angle deposition (GLAD) or plasma treatment of a nanosphere lithography (NSL) mask prior to the deposition of a thin Au film. The nanohole arrays obtained with a 450 nm nanospheres mask are characterized using atomic force microscopy (AFM) to determine the depth and the width of the nanoholes, and the periodicity of the nanohole arrays. The analytical properties are reported in terms of the surface plasmon (SP) excitation wavelength (500 nm to 1000 nm), sensitivity to refractive index (27 nm RIU(-1) to 487 nm RIU(-1)), sensitivity to monolayer formation (shift of the SP band by approx. 1 nm), and refractive index resolution (10(-4) RIU). These simple techniques produce well-ordered nanohole arrays with tunable analytical and physical properties for the development of biosensors.