In this work, two forms of keratins, kerateine (KR) and keratose (KO), were fabricated respectively into electrospun nanofibers by combination with polyurethane (PU). The differences of the structure and material properties between KR and KO based fibers were investigated by SEM observation, ATR-FTIR, XRD, contact angle, tensile test, in vitro degradation and cytocompatibility assay. The results indicated that the KR based nanofibers exhibited a higher tensile modulus, lower fracture strain and slower degradation rate, mainly due to the reformation of disulfide crosslinking between the regenerated cysteines in KR after the reductive extraction. The KO based nanofibers demonstrated a stronger hydrophilic property and higher water uptake ability due to the cysteic acid residues resulting from the oxidative extraction. Furthermore, the combination of keratins, regardless of KR or KO, could obviously improve the cytocompatibility of PU, especially in the cell attachment stage.