Pars plana vitrectomy plays an important role in treating serious ophthalmic diseases, which requires subsequent substitution of the natural vitreous. However, the current substitutes are known for the drawbacks in instability, toxicity, and injection‐induced fragmentation, so there is a very urgent need in novel artificial vitreous substitute for clinical practice. In this study, DNA supramolecular hydrogel is evaluated as a potential artificial vitreous substitute. It is observed that DNA supramolecular hydrogel exhibits relatively strong mechanical strength, shear‐thinning behavior, rapid recoverability, and other physical properties similar to the human vitreous body. The in vivo experiments further prove the injectability, excellent biocompatibility, and in vivo stability. With the support of the DNA supramolecular hydrogel, the morphology and basic functions of the operated eyes are well recovered without obvious inflammation, which demonstrate the DNA supramolecular hydrogel shows enormous potential as vitreous substitute.