During the last two decades, scientists have developed various methods that allow the detection and manipulation of single molecules, which have also been called “in singulo” approaches. Fundamental understanding of biochemical reactions, folding of biomolecules, and the screening of drugs were achieved by using these methods. Single-molecule analysis was also performed in the field of DNA nanotechnology, mainly by using atomic force microscopy. However, until recently, the approaches used commonly in nanotechnology adopted structures with a dimension of 10–20 nm, which is not suitable for many applications. The recent development of scaffolded DNA origami by Rothemund made it possible for the construction of larger defined assemblies. One of the most salient features of the origami method is the precise addressability of the structures formed: Each staple can serve as an attachment point for different kinds of nanoobjects. Thus, the method is suitable for the precise positioning of various functionalities and for the single-molecule analysis of many chemical and biochemical processes. Here we summarize recent progress in the area of single-molecule analysis using DNA origami and discuss the future directions of this research.