In this study, we investigated whether the nanofibers produced by natural-synthetic polymers can probably promote the proliferation of co-cultured adipose-derived stem cells/human fibroblast cells (ADSs/HFCs) and synthesis of collagen. Nanofiber was fabricated by blending gelatin and poly (L-lactide co-ɛ-caprolactone) (PLCL) polymer nanofiber (Gel/PLCL). Cell morphology and the interaction between cells and Gel/PLCL nanofiber were evaluated by FESEM and fluorescent microscopy. MTS assay and quantitative real-time polymerase chain reaction were applied to assess the proliferation of co-cultured ADSs/HFCs and the collagen type I and III synthesis, respectively. The concentrations of two cytokines including fibroblast growth factor-basic and transforming growth factor-β1 were also measured in culture medium of co-cultured ADSs/HDCs using enzyme-linked immunosorbent assay assay. Actually, nanofibers exhibited proper structural properties in terms of stability in cell proliferation and toxicity analysis processes. Gel/PLCL nanofiber promoted the growth and the adhesion of HFCs. Our results showed in contact co-culture of ADSs/HFCs on the Gel/PLCL nanofiber increased cellular adhesion and proliferation synergistically compared to non-coated plate. Also, synthesis of collagen and cytokines secretion of co-cultured ADSs/HFCs on Gel/PLCL scaffolds is significantly higher than non-coated plates. To conclude, the results suggest that Gel/PLCL nanofiber can imitate physiological characteristics in vivo and enhance the efficacy of co-cultured ADSs/HFCs in wound healing process.