This Review focuses on how to use DNA nanostructures as scaffolds to organize biological molecules. First, we introduce the use of structural DNA nanotechnology to engineer rationally designed nanostructures. Second, we survey approaches used to generate protein-DNA conjugates. Third, we discuss studies exploring DNA scaffolds to create DNA nanodevices to analyze protein structures, to engineer enzyme pathways, to create artificial light-harvesting systems, and to generate nanomachines in vitro and in vivo. Future challenges and perspectives of using DNA nanostructures as programmable biomolecular scaffolds are addressed at the end.