DNA is a perfect polymeric molecule for interfacing biology with material science to construct hydrogels that represent fascinating properties for a wide variety of biomedical applications. Tunable multifunctionality, convenient programmability, adequate biocompatibility, biodegradability, capability of precise molecular recognition, and high versatility have made DNA an irreplaceable building block for the construction of novel 3D hydrogels. DNA can be used as the only component of a hydrogel, the backbone or a cross-linker that connects the main building blocks to form hybrid hydrogels through chemical reactions or physical entanglement. Responsive constructs of DNA with superior mechanical properties are very commonly reported nowadays, which can undergo macroscopic changes induced by various triggers, including alteration in ionic strength, temperature, and pH. These hydrogels can be prepared by various types of DNA building blocks, such as branched double-stranded DNA, single-stranded DNA, X-shaped DNA, or Y-shaped DNA through intermolecular i-motif structures, DNA hybridization, enzyme ligation, or enzyme polymerization. These hydrogels are envisioned for a variety of applications, such as drug delivery, sensing, tissue engineering, 3D cell culture, and providing template for nanoparticle synthesis.