Nucleic acids have enabled the fabrication of self-assemblies and dynamic operations. Among different functional nucleic acids, aptamers can specifically bind to a wide range of targets, including proteins, viral antigens, living cells and even tissues, and have thus emerged as molecular recognition tools in molecular medicine. Hence, aptamer-functionalized nucleic acid nanotechnology offers applications of biosensing, bioimaging, and cancer therapy. In this review, after a brief overview of nucleic acid nanotechnology, we focus on the integration of aptamers with nucleic acid nanotechnology, including self-assembly constructions and dynamic molecular manipulations. The emerging applications in molecular medicine are subsequently reviewed with aptamer-based self-assemblies and aptamer-involved dynamic molecular manipulation. For convenience, applications are broadly categorized into biosensing, bioimaging, and cancer therapy. Finally, challenges and potential development of nucleic acid nanotechnology are discussed.