Nucleic acid‐based hydrogels that integrate intrinsic biological properties of nucleic acids and mechanical behavior of their advanced assemblies are appealing bioanalysis and biomedical studies for the development of new‐generation smart biomaterials. It is inseparable from development and incorporation of novel structural and functional units. This review highlights different functional units of nucleic acids, polymers, and novel nanomaterials in the order of structures, properties, and functions, and their assembly strategies for the fabrication of nucleic acid‐based hydrogels. Also, recent advances in the design of multifunctional and stimuli‐responsive nucleic acid‐based hydrogels in bioanalysis and biomedical science are discussed, focusing on the applications of customized hydrogels for emerging directions, including 3D cell cultivation and 3D bioprinting. Finally, the key challenge and future perspectives are outlined.