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Over the past few decades, DNA nanotechnology engenders a vast variety of programmable nanostructures utilizing Watson–Crick base pairing. Due to their precise engineering, unprecedented programmability, and intrinsic biocompatibility, DNA nanostructures cannot only interact with small molecules, nucleic acids, proteins, viruses, and cancer cells, but also can serve as nanocarriers to deliver different therapeutic agents. Such addressability innate to DNA nanostructures enables their use in various fields of biomedical applications such as biosensors and cancer therapy. This review is begun with a brief introduction of the development of DNA nanotechnology, followed by a summary of recent applications of DNA nanostructures in biosensors and therapeutics. Finally, challenges and opportunities for practical applications of DNA nanotechnology are discussed.