Keratin is widely used in the biomaterial application, but the keratin prepared by the physical or chemical approach has relatively low molecular weight and mechanical properties. Here we report the preparation of high molecular keratin (HMK) with molecular weight of 120 kDa via multi-enzyme cascade pathway and its application in wound healing. Briefly, we prepared the soluble keratin from wool by keratinase and improved the molecular weight of keratin by transglutaminase (TGase). The HMK was coelectrospun with poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and the prepared nanofibrous mats demonstrated improved mechanical properties. Ag nanoparticles (AgNPs) were synthesized on the nanofibers via in situ bioreduction, using the above-mentioned keratinase as the reducing agent. It is demonstrated that the PHBV/HMK/AgNPs nanofibrous mats possess favorable antibacterial properties and good biocompatibility. Moreover, in vivo wound healing assessment, the PHBV/HMK/AgNPs membrane displayed better wound healing ability than the control group. These results indicate that PHBV/HMK/AgNPs mats exhibit significant potential in tissue engineering.