DNA origami technique was first introduced in 2006 by Rothemund, it has gained widespread research interest and led to explosive achievements, in which long single-stranded DNA (ssDNA) is folded into a designed nanostructure, in either two dimensions (2D) or three dimensions (3D), with the aid of many shorter staple strands. A series of methods for new design principles for DNA origami nanostructures have already been proposed, ranging from the preparation of scaffold to the folding mechanisms. After that, novel strategies in functionalizing DNA origami nanostructures and their practical applications are gradually becoming research hotspots. This review focuses on the development in the new scaffold design approaches, folding conditions, various nano objects functionalized on DNA origami nanostructures, and their applications as drug carriers in the recent five years. We anticipate more exploratory and extendible work developed based on the summary of progress obtained previously.