Electrical stimulation with conductive biomaterials shows great promising to promote skin wound healing in practical application. In this study, polypyrrole@keratin nanofiber membrane (PPy@KNM) with good conductivity was prepared using electrospinning technology and chemical in situ polymerization of pyrrole monomers. Morphological, chemical, X-ray and surface analyses suggested that PPy was deposited on the keratin nanofiber membrane (KNM). The PPy@KNM with the highest conductivity and the largest tensile strength was prepared with an oxidant concentration of 0.6 M and a dopant concentration of 0.5 M of the p-toluenesulfonic acid (p-TSA). In vitro cell culture experiments revealed that the conductive PPy@KNM performed good biocompatibility and no cytotoxicity. Electrical stimulation via the PPy@KNM can reasonably accelerate cell growth and cell proliferation with an optimal current intensity of 80 μA and a stimulation time of 5 min/d. The research outcomes demonstrated that PPy@KNM is suitable to be applied as a functional biomedical material to promote wound healing.