A hydrogel has been developed into a promising scaffold for cell encapsulation or drug delivery useful in various biorelated research areas. DNA, with its outstanding specificity and flexibility and intrinsic biocompatibility, has been utilized as a new building block in the construction of this new three dimensional material-a DNA based hydrogel. Here, we report the synthesis, properties and applications of a DNA based hydrogel system. The DNA hydrogels are entirely constructed from branched DNA building blocks such as X-shaped DNA (X-DNA), Y-shaped DNA (Y-DNA), and T-shaped DNA (T-DNA) through an enzyme-catalyzed reaction. These DNA hydrogels can be easily patterned into different sizes with millimeter or micrometer scale and different shapes including cylindrical, rectangular, cross, star, and even the “CORNELL” logo. By adjusting the initial concentrations and original shapes of the different building blocks, the external and internal morphology and chemical/physical properties of the DNA hydrogel were easily tuned: The X-DNA based hydrogel (X-DNA gel) showed both the most swelling degree and mechanical strength as well as the higher resistance to chemical and biological degradation. Inspired by these characteristics of the DNA hydrogels, we have developed them into a drug delivery system, an artificial extracellular matrix, and even a cell-free protein producing template.