We report an effective method to fabricate two-dimensional (2D) periodic oxide nanopatterns using S-layer proteins as a template. Specifically, S-layer proteins with a unit cell dimension of 20 nm were reassembled on silicon substrate to form 2D arrays with ordered pores of nearly identical sizes (9 nm). Octadecyltrichlorosilane (ODTS) was utilized to selectively react with the S-layer proteins, but not the Si surface exposed through the pores defined by the proteins. Because of the different surface functional groups on the ODTS-modified S-layer proteins and Si surface, area-selective atomic layer deposition of metal oxide-based high-k materials, such as hafnium oxide, in the pores was achieved. The periodic metal oxide nanopatterns were generated on Si substrate after selective removal of the ODTS-modified S-layer proteins. These nanopatterns of high-k materials are expected to facilitate further downscaling of logic and memory nanoelectronic devices.