Diabetic skin ulcers are a major cause of disability, yet effective treatments remain limited due to microvascular dysfunction. This study investigated the effects of a glycol chitosan-silk fibroin hydrogel loaded with Ephrin-B2 (GCS-SF-B2 Gel) on the healing of full-thickness skin wounds in a diabetic mouse model. We prepared the hydrogel by mixing glycol chitosan and silk fibroin solutions, followed by lyophilization and adsorption of Ephrin-B2. The GCS-SF-B2 Gel exhibited a porous structure with a swelling capacity of up to 1600% and a moisturizing duration of up to 12 h. In vitro assays demonstrated that the hydrogel significantly promoted the proliferation and migration of NIH 3T3 cells. In a diabetic mouse model with full-thickness skin defects, the GCS-SF-B2 Gel demonstrated superior therapeutic efficacy compared to control, evidenced by macroscopic healing, increased fibroblast accumulation, improved collagen deposition, and significant angiogenesis at the wound site. These findings suggest that GCS-SF-B2 Gel is a promising candidate for promoting the healing of refractory diabetic wounds.