The Wharton's jelly (WJ) contains significant amounts of extracellular matrix (ECM) components and rich source of endogenous growth factors. In this study, we designed a new biomimetic spongy scaffold from decellularized WJ-derived ECM and used it as a skin substitute. Histological analysis and biochemical assays showed that bio-active molecules preserved in the fabricated scaffolds and that the scaffolds have highly interconnected porous structure. Cytotoxicity and mechanical evaluation of the scaffold indicated that it is non-toxic and has appropriate mechanical properties. MTT assay, SEM and histological analysis of human fibroblast, seeded on the scaffolds, confirmed cellular viability, attachment, penetration and proliferation. The effectiveness of WJ-derived scaffolds in the regeneration of full-thickness wound was assessed through an in vivo experiment. Our results demonstrated that the scaffolds were well integrated into the mouse tissue and absorbed the exudates after one week. Unlike the controls, in WJ group there were not only complete wound closing and disappearance of the scab, but also complete reepithelialization, newly generated epidermal layers and appendages after 12 days of implantation. Taken together, our results indicate that WJ-derived scaffolds are able to improve attachment, penetration and growth of the fibroblast cells and speed up the healing processes, which would offer a proper skin graft for wound healing.