Chirality is a universal phenomenon in nature. Chiral structures refer to two objects that are mirror images and cannot be superimposed on each other by any kind of translation or rotation. Nucleic acids, including DNA and RNA, are chiral structures. Different chiral geometries of nucleic acids, such as A-form, B-form, and Z-form DNA, and mirror L-Nucleic acids, have different properties and physiological functions. This review covers the fundamentals and recent progress in nucleic acid-based chiral nanostructures and their biomedical applications. We begin by introducing chiral geometries of nucleic acids, including naturally occurring A-form, B-form, Z-form DNA and artificially synthesized mirror L-nucleic acids. Then the recent advances in creating chiral nanostructures using nucleic acids themselves are presented in the following part. In particular, we highlight the emerging biomedical applications of nucleic acids-based chiral nanostructures. Finally, in the conclusion section, we provide our views on future challenges and prospects of nucleic acid-based chiral nanostructures.