Mica is the current substrate of choice for DNA nanostructure imaging, mainly due to its atomically flat surface. However, these mica substrates are often not optically clear. In this work, sapphire has been evaluated as an alternative substrate, with potential to enable parallel optical and AFM studies. Well known for its thermal and chemical properties, sapphire is a hard ionic material with excellent optical properties. Because sapphire lacks the excellent basal cleavage properties of the sheet silicate mica, a process to anneal it at high temperature in water vapor was developed to achieve near atomically smooth (average roughness = 0.141 nm) terraces. AFM imaging was used to determine the dimensions of these terraces and to characterize the morphology of the DNA nanostructures, revealing that their structures were preserved, indicating that annealed c-plane cut (0001) sapphire is a promising substitute for mica as a flat and transparent substrate for DNA nanostructure studies.