The Watson–Crick complementarity of DNA is exploited to construct periodically patterned nanostructures, and we herein demonstrate tile-based three dimensional (3D) multilayered DNA nanostructures that incorporate two design strategies: vertical growth and horizontal layer stacking with substrate-assisted growth. To this end, we have designed a periodically holed double–double crossover (DDX) template that can be used to examine the growth of the multilayer structures in both the vertical and horizontal directions. For vertical growth, the traditional 2D double crossover (DX) DNA lattice is seeded and grown vertically from periodic holes in the DDX template. For horizontal stacking, the DDX layers are stacked by binding the connector tiles between each layer. Although both types of multilayers exhibited successful formation, the observations with an atomic force microscope indicated that the DDX layer growth achieved with the horizontal stacking approach could be considered to be slightly better relative to the vertical growth of the DX layers in terms of uniformity, layer size, and discreteness. In particular, the newly designed DDX template layer provided a parallel arrangement between each domain with substrate-assisted growth. This kind of layer arrangement suggests a possibility of using our design scheme in the construction of other periodic structures.