Abstract: DNA hydrogels hold great potential for biological and biomedical applications owing to their programmable nature and macroscopic sizes. However, most previous studies involve spontaneous and homogenous gelation procedures in solution, which often lack precise control. A clamped hybridization chain reaction (C-HCR)-based strategy has been developed to guide DNA self-assembly to form macroscopic hydrogels. Analogous to catalysts in chemical synthesis or seeds in crystal growth, we introduced DNA initiators to induce the gelation process, including crosslinked self-assembly and clamped hybridization in three dimensions with spatial and temporal control. The formed hydrogels show superior mechanical properties. The use of printed, surface-confined DNA initiators was also demonstrated for fabricating 2D hydrogel patterns without relying on external confinements. This simple method can be used to construct DNA hydrogels with defined geometry, composition, and order for various bioapplications.