Electrospinning is a versatile technique to make biomimetic and nanostructured scaffolds for skin tissue engineering. In this study we have electrospun and characterized chitosan (C)-poly(vinyl alcohol) (PVA) blend nanofibers as dermal substitutes and compared with 2D C-PVA films. The in vitro characterization of the C-PVA nanofibers and 2D films were evaluated using mouse 3T3 fibroblast cells and our results demonstrated that the cells adhered and proliferated on the surface of C-PVA nanofibers. In our animal studies, the implantation of C-PVA nanofibers along with topical administration of growth factor R-Spondin 1 on full thickness wounds created on rats showed 98.6% wound closure after two weeks post-surgery. The catalase and superoxide dismutase activity of the healing tissue was significantly higher in the groups treated with topical administration of growth factor and C-PVA nanofibers (p < 0.05). Thus these C-PVA nanofibers along with novel growth factor are promising new biomaterials that could be used as dermal substitutes for accelerated wound healing.