Silk fibroin (SF) has been broadly applied in wound dressing fabrication because of its proper features for wound healing. In this work, we developed a carboxymethyl cellulose (CMC)/gelatin blend film with different concentrations of glycerol, and modified the optimized film with an SF layer through electrospinning process. Tensile strength and cell viability evaluation of blend films demonstrated that the glycerol content of 3% could be suitable as the substrate layer for the two‐layer wound dressing. The morphology of the blend film and electrospun nanofibers was obtained from scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). It concluded that structural changes had been occurred in both layers after cross‐linking with glutaraldehyde vapor. Further, it was shown that the mechanical properties of the two‐layer enhanced with the addition of SF. Moreover, the swelling ratio was higher than those of SF itself, due to the hydrophilic property of CMC/gelatin blend film. The biocompatibility of fibroblasts was investigated by MTT assay, and the coating showed an improvement in cell proliferation because of the cytocompatibility nature of SF. All results suggest that the prepared wound dressing could be a desirable candidate for wound healing applications.