Abstract A rapidly formed supramolecular polypeptide–DNA hydrogel was prepared and used for in situ multilayer three‐dimensional bioprinting for the first time. By alternative deposition of two complementary bio‐inks, designed structures can be printed. Based on their healing properties and high mechanical strengths, the printed structures are geometrically uniform without boundaries and can keep their shapes up to the millimeter scale without collapse. 3D cell printing was demonstrated to fabricate live‐cell‐containing structures with normal cellular functions. Together with the unique properties of biocompatibility, permeability, and biodegradability, the hydrogel becomes an ideal biomaterial for 3D bioprinting to produce designable 3D constructs for applications in tissue engineering.

Rapid Formation of a Supramolecular Polypeptide–DNA Hydrogel for In Situ Three‐Dimensional Multilayer Bioprinting