| Summary: | Abstract DNA has been used as a robust material for the building of a variety of nanoscale structures and devices owing to its unique properties. Structural DNA nanotechnology has reported a wide range of applications including computing, photonics, synthetic biology, biosensing, bioimaging, and therapeutic delivery, among others. Nevertheless, the foundational goal of structural DNA nanotechnology is exploiting DNA molecules to build three‐dimensional crystals as periodic molecular scaffolds to precisely align, obtain, or collect desired guest molecules. Over the past 30 years, a series of 3D DNA crystals have been rationally designed and developed. This review aims to showcase various 3D DNA crystals, their design, optimization, applications, and the crystallization conditions utilized. Additionally, the history of nucleic acid crystallography and potential future directions for 3D DNA crystals in the era of nanotechnology are discussed.
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