AbstractHere, the Fenton reaction is used to prepare silk hydrogels through oxidation of tyrosine residues in silk fibroin, leading to dityrosine crosslinking. At pH 5.7, gelation occurs rapidly within 30 s, and the resultant opaque gels show soft properties with a storage modulus of ≈100 Pa. The addition of ascorbic acid to the Fenton reaction increases the dityrosine bonds in the hydrogels but has little effect on the rheological or mechanical properties. The results indicate that Fe(III) ions significantly interacted with silk fibroin during the Fenton reaction, most likely binding to sites such as tyrosine, glutamate, and aspartate residues, triggering the formation of β‐sheet structures that may impede dityrosine bond formation due to steric hindrance. The use of an iron chelator or the operation of the Fenton reaction at pH 9.2 enables control over the interaction of Fe(III) ions with silk fibroin, achieving a hydrogel with improved optical properties and enhanced dityrosine bond formation. Hydrogels prepared by the Fenton reaction are cytocompatible as L929 mouse fibroblasts remain viable and are proliferative when seeded on the hydrogels. The results offer a useful approach to generate chemically crosslinked silk fibroin hydrogels without the use of enzyme‐catalyzed reactions for biomedical applications.