This study focuses on preparation and structural investigation of the water insoluble Keratin/poly (ethylene oxide) (PEO) blend nanofiber mat with high content of keratin for biomedical utilization. A modified secondary crosslinking process in oxygen atmosphere was first employed to improve the water insolubility of the electrospun keratin/PEO nanofibers with high keratin content, following the primary crosslinking process with ethylene glycol diglycidyl ether (EGDE), for cell culture. A systematic quantitative analysis of liquid viscosity, FTIR, XRD, and TG were conducted to illustrate the electrospinnability of the primary crosslinked keratin/PEO blend water solution and the water insolubility of the secondary crosslinked nanofiber mat. The results indicated that the increase of the keratin molecular weight by the primary crosslinking reaction between keratin molecules by EGDE might be the main reason for the improved electrospinnability of the solution. The water insolubility of the secondary crosslinked nanofiber mat was attributed to the rebuilt of the sulfur crosslinking bonds between keratin formed in pure oxygen atmosphere during the secondary crosslinking process. The biocompatibility of the water insoluble keratin/PEO blend nanofiber mat was also investigated with cell cultivation, suggesting the nanofibers had a potential in tissue engineering and biomaterials field. (C) 2016 Elsevier Ltd. All rights reserved.