From being genetic material to being exploited as an intelligent biomaterial, DNA has traveled a lot of scientific space of research and innovation. DNA can assemble into macromolecular polymeric networks based on sequences or by physically cross-linking their bulky lengthy strands. DNA is a polyanionic, hydrophilic, and polyelectrolytic natural biomaterial that can absorb large amounts of water mostly via H-bond interactions. The ability of DNA to attract water enables it form DNA-based hydrogels. DNA hydrogels offer many desirable qualities, making them an ideal choice as a desirable biomaterial for diverse applications. DNA Hydrogels show biodegradability, biocompatibility, modularity, non-toxicity, hydrophilicity, self-healing ability, and the ability to probe, program, and reprogram diverse biological systems. This chapter focuses on pure DNA-based hydrogels, their principles, and synthesis methods. We outlay various characterization tools and techniques followed by their biological applications and brief conclusion about their future employability for diverse biomedical applications.