Nucleic acid probes have the advantages of excellent biocompatibility, biodegradability, versatile functionalities and remarkable programmability. However, the low biostability of nucleic acid probes under complex physiological conditions limits their in vivo application. Despite impressive progress in the development of inorganic material-mediated biostable nucleic acid nanostructures, uncertain systemic toxicity of composite nanocarriers has hindered their application in living organisms. In the field of biomedicine, as a promising alternative capable of avoiding potential cytotoxicity, biologically stable nanostructures composed entirely of DNA oligonucleotides have been rapidly developed in recent years, offering an exciting in vivo tool for cancer diagnosis and clinical treatment. In this review, we summarize the recent advances in the development of nuclease-resistant DNA nanostructures with different geometrical shapes, such as tetrahedron, octahedron, DNA triangular prism (DTP), DNA nanotubes and DNA origami, introduce innovative assembly strategies, and discuss unique structural advantages and especially biological applications in cellular imaging and targeted drug delivery in an organism. Finally, we conclude with the challenges in the clinical development of DNA nanostructures and present an outlook of the future of this rapidly expanding field.