Since its discovery in 2006, the DNA origami technique has revolutionized bottom-up nanofabrication. This technique is simple yet versatile and enables the fabrication of nanostructures of almost arbitrary shapes. Furthermore, due to their intrinsic addressability, DNA origami structures can serve as templates for the arrangement of various nanoscale components (small molecules, proteins, nanoparticles, etc.) with controlled stoichiometry and nanometer-scale precision, which is often beyond the reach of other nanofabrication techniques. Despite the multiple benefits of the DNA origami technique, its applicability is often restricted by the limited stability in application-specific conditions. This Review provides an overview of the strategies that have been developed to improve the stability of DNA-origami-based assemblies for potential biomedical, nanofabrication, and other applications.