High‐content keratin/poly (ethylene oxide) (PEO) (90/10) blend nanofibers were prepared by electrospinning combined with a two‐step cross‐linking process. The keratin/PEO aqueous solution was firstly mixed with ethylene glycol diglycidyl ether (EGDE) as cross‐linker and then electrospun into nanofibers. The resulting nanofibrous mats were cross‐linked with EGDE vapor to decrease the solubility of nanofibers in water. The morphologies and properties of electrospun fibers were investigated by SEM, FTIR, TG, XRD, and contact angle testing, respectively. The results showed that the morphologies of nanofibers were uniform at the fiber average diameter of 300 nm with negligible bead defects by adding EGDE to keratin/PEO solutions. The cross‐linking results showed that EGDE vapor could improve the hydrophobic property of blended nanofibers. The crystallinity of the keratin/PEO blend nanofiber mat increased from 13.14% for the uncross‐linked sample to 21.54% and 35.15% for the first cross‐linked and second cross‐linked samples, respectively. Free defect nanofiber mats with high keratin content producing from this two‐step cross‐linking process are particularly promising for tissue engineering and cell‐seeded scaffold.