Mitochondria, one of the most important organelles, represent a crucial subcellular target for fundamental research and biomedical applications. Despite significant advances in the design of DNA nanotechnologies for a variety of bio‐applications, the dearth of strategies that enable mitochondria targeting for subcellular molecular imaging and therapy remains an outstanding challenge in this field. In this Minireview, we summarize the recent progresses on the emerging design and application of DNA nanotechnology for mitochondria‐targeted molecular imaging and tumor treatment. We first highlight the engineering of mitochondria‐localized DNA nanosensors for in situ detection and imaging of diverse key molecules that are essential to maintain mitochondrial functions, including mitochondrial DNA and microRNA, enzymes, small molecules, and metal ions. Then, we compile the developments of DNA nanotechnologies for mitochondria‐targeted anti‐tumor therapy, including modularly designed DNA nanodevices for subcellular delivery of therapeutic agents, and programmed DNA assembly for mitochondrial interference. We will place an emphasis on clarification of the chemical principles of how DNA nanobiotechnology can be designed to target mitochondria for various biomedical applications. Finally, the remaining challenges and future directions in this emerging field will be discussed, hoping to inspire further development of advanced DNA toolkits for both academic and clinical research regarding mitochondria.