As one of the most interesting II–IV compound semiconductors, ZnO has large band gap (3.37 eV) and high excitonic binding energy (60 meV). Based on this, it has attracted a great deal of attention for applications in ultraviolet light-emitting devices (LED) and photodetectors. There are many preparation methods to prepare ZnO films, such as metal organic chemical vapor deposition (MOCVD), magnetron sputtering, vacuum thermal evaporation, and so on. Among them, there are many advantages on using magnetron sputtering to form ZnO thin films, such as good adhesion, good thickness uniformity, high density of films, so we take advantage of this method in our experiment. In this work, we present a simple, rapid and cost effective method to fabricate ordered periodic substrates by preparing single layer polystyrene microspheres masks, with 300, 600, 800and 1100 nm in diameters. Then the layer of zinc oxide thin films on the mask by RF magnetron sputtering technique have been deposited, and two-dimensional zinc oxide nano-array samples were obtained at last. Using this active plasmonic substrate, the optical properties of ZnO films on polystyrene microspheres template has been investigated.