Interfacial assembly has been intensively investigated in fabricating biomaterials and nanodevices for various applications. Recently, due to the precise sequence programmability, unique molecular recognition ability, and good biocompatibility, deoxyribonucleic acid (DNA) has been explored as superior building blocks to assemble at bio-interface for manipulating biological entities. To the best of our knowledge, the advances in this area have not been systematically summarized. To provide an overview of the area, in this review, the recently developed DNA assembly strategies on bio-interfaces were well summarized, and their representative works are exampled to illustrate how to rationally and elaborately design DNA molecules to realize functional integration and emerging of novel biological functionalities with high controllability and programmability. Furthermore, the biomedical applications of DNA assembly at bio-interface are categorially elaborated. The fascinating and unique advantages of DNA assembly systems are fully discussed in the exemplified applications to show the distinguished perspective of DNA in the future development. At the end of this review, the current limitations and challenges in applications and potential improvement strategies for DNA assembly at bio-interface are fully discussed. The future development direction is deliberated. We envision that this review will help scientists in the interdisciplinary fields gain a more comprehensive understanding of the DNA assembly at bio-interface, and therefore jointly promote the advances in this field.