Autologous skin grafting, the standard treatment for severe burns, is sometimes not possible due to the limited available skin surfaces for the procedure. With advances in tissue engineering, various cell-based skin substitutes have been developed to serve as skin replacements and to promote tissue regeneration and healing. In this work, we propose the use of cell sheet technology to fabricate keratinocyte-fibroblast tissue constructs from the temperature-responsive poly(N-isoproprylacrylamide-co-acrylamide) (PNIAM-co-AM) grafted surfaces for the treatment of burn wounds. The characteristics of the human keratinocyte and fibroblast cell sheets harvested using PNIAM-co-AM grafted surfaces were similar to those cell sheets detached from the commercially-available UpCellTM plates. Upon lowering the incubation temperature, confluent keratinocytes and fibroblasts could be detached as intact sheets, consisting of biologically active cells, as indicated by their high cell viability and their reattachment, migratory, and proliferative activities. A histological analysis of the stratified keratinocyte-fibroblast cell sheets revealed the evidence of cell migration and tissue reorganization to form two distinct epidermal and dermal layers, quite similar to the skin tissue's structure. In addition, the keratinocyte-fibroblast sheets could synthesize and release significant amounts of essential cytokines and growth factors involved in regulating the wound healing process, including IL-1α, IL-6, TNF-α, VEGF, and bFGF, implying the therapeutic effect of these cell sheets, which could be beneficial to accelerate tissue repair and regeneration, leading to faster wound healing.