Because of the defined double-helical structure and programmed nature, DNA is an excellent molecule for the creation of various multidimensional nanostructures and the programmed placement of functional molecules and materials. DNA origami is an emerging strategy in the DNA nanotechnology field for designing defined two-dimensional DNA nanostructures. In this review, we focus on and describe the following DNA origami-based researches; (i) DNA origami-templated molecular assembly, (ii) programmed DNA origami assembly, (iii) design and construction of various three-dimensional DNA origami structures, (iv) programmed functionalization of DNA origami and combination with top-down nanotechnology, (v) single molecular observation on designed DNA origami, and (vi) DNA nanomachines working on DNA origami.