Hypoxia plays a critical role in the development and wound healing process, as well as a number of pathological conditions. Here, dextran-based hypoxia-inducible (Dex-HI) hydrogels formed with in situ oxygen consumption via a laccase-medicated reaction are reported. Oxygen levels and gradients were accurately predicted by mathematical simulation. It is demonstrated that Dex-HI hydrogels provide prolonged hypoxic conditions up to 12 h. The Dex-HI hydrogel offers an innovative approach to delineate not only the mechanism by which hypoxia regulates cellular responses, but may facilitate the discovery of new pathways involved in the generation of hypoxic and oxygen gradient environments.