Tissue-engineered skin substitutes provided a feasibility to overcome the shortage of skin autograft by culturing keratinocytes and dermal fibroblasts in vitro. In this study, we applied bi-layer gelatin-chondrointin-6-sulfate-hyaluronic acid (gelatin-C6S-HA) biomatrices onto the severe combined immunodeficiency (SCID) mice to evaluate its effect on promoting wound healing. Human foreskin keratinocytes and dermal fibroblasts were cultured with reconstructed skin equivalent (rSE) for 7 days. The rSE was then grafted to the dorsum of SCID mice to evaluate its biocompatibility by histologic and immunohistochemistry analysis. The results showed that human epidermis were well-developed with the expression of differentiated markers and basement membrane-specific proteins at 4 weeks. After implantation, the percentages of skin graft take were satisfactory, while cell-seeded group was better than non-cell-seeded one. The basement membrane proteins including laminin, type IV collagen, type VII collagen, integrin alpha6, and integrin beta4 were all detected at the dermal-epidermal junction, which showed a continuous structure in the 4 weeks after grafting. This bi-layer gelatin-C6S-HA skin substitute not only has positive effect on promoting wound healing, but also has high rate of graft take. This rSE would have the potential to be applied on the extensively and deeply burned patients who suffer from severe skin defect in the near future.