Nucleic acid nanotechnology has been developed to be a promising strategy to construct various nano-biomaterials with structural programmability, spatial addressability, and excellent biocompatibility. Self-assembled nucleic acid nanostructures have been employed in a variety of biomedical applications, such as bio-imaging, diagnosis, and therapeutics. In this manuscript, we will review recent progress in the development of multifunctional nucleic acid nanostructures as gene drug delivery vehicles. Therapeutic systems based on RNA interference (RNAi), clustered regularly interspaced short palindromic repeat associated proteins 9 system (CRISPR/Cas9) genome editing, gene expression, and CpG-based immunostimulation will be highlighted. We will also discuss the challenges and future directions of nucleic acid nanotechnology in biomedical research.