Display omitted •The first use of a macromer cross-linker for DNA gelation.•The first use of isocyanates for DNA cross-linking.•The cross-linking of DNA molecules without a catalyst.•The elasticity of DNA gels has been greatly increased by the DNA renaturation. Denatured DNA molecules were used as building blocks to fabricate thermoresponsive hydrogels by cross-linking DNA strands with an isocyanate (NCO) end-capped star-shaped poly(EO-stat-PO) cross-linker. The NCO groups reacted with the amine groups of DNA’s bases through urea links while base pairing between the matching strands of DNA led to the self-assembly of DNA into a three-dimensional hydrogel network. Analysis of the ultrastructure by scanning force microscopy (SFM) revealed the temperature-mediated transition from the rod-to-coil structure as a result of the destabilization of double-stranded DNA (dsDNA) to form a single-stranded DNA (ssDNA), which eventually resulted in a DNA network due to base pairing, along with hairpin loops due to mismatches between nucleotides. Dynamic oscillatory and large deformation creep tests, as well as stress relaxation tests, revealed the formation of robust cross-linked gels: A ten-fold rise in the elastic modulus was observed for the covalent DNA gel compared to the physical gel at the identical DNA concentration. The thermoresponsiveness of the hydrogels was confirmed by a heating–cooling cycle while the non-porous internal morphology of the hydrogel was demonstrated through scanning electron microscopic analysis. Overall, this is the first study showing the fabrication of covalent DNA hydrogels using an NCO-end-capped hydrophilic prepolymer. Such gels have a high potential to be exploited in many fields to benefit from the structural properties of DNA molecules.