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In situcross-linked hydrogels have the advantage of effectively fulfilling the wound in its shape and depth. Amongst the new generation of natural-based biopolymers being proposed for wound care and skin regeneration, silk sericin is particularly interesting due to its exceptional properties such as biocompatibility, biodegradability, and antioxidant behavior, among others. In this study, a new enzyme-mediated cross-linked hydrogel composed of silk sericin is proposed for the first time. The developed hydrogel cross-linking strategy was performed via horseradish peroxidase, under physiological conditions, and presented gelling kinetics under 3 min, as demonstrated by its rheological behavior. The hydrogels presented a high degree of transparency, mainly due to their amorphous conformation. Degradation studies revealed that the hydrogels were stable in phosphate buffer solution (PBS) (pH 7.4) for 17 days, while in the presence of protease XIV (3.5 U/mg) and under acute and chronic physiological pH values, the stability decreased to 7 and 4 days, respectively. During protease degradation, the present sericin hydrogels demonstrated antioxidant activity. In vitrostudies using an L929 fibroblast cell line demonstrated that these hydrogels were noncytotoxic, promoting cell adhesion and massive cell colonization after 7 days of culture, demonstrating that cells maintained their viability and proliferation. In addition, the application of sericin-based hydrogel in an in vivodiabetic wound model validated the feasibility of the in situmethodology and demonstrated a local anti-inflammatory effect, promoting the healing process. This study presents a simple, fast, and practical in situapproach to produce a sericin-based hydrogel able to be applied in low exudative chronic wounds. Moreover, the study herein reported fosters the valorization of a textile industrial by-product by its integration in the biomedical field.