Post-crosslinking treatment is essential for improving the water tolerance of the electrospinning keratin/Poly(ethylene oxide)(PEO) nanofiber membrane to meet the requirement of the biomedical application. In this study, three ecofriendly low-costmethods, hot air, pure oxygen, and ultraviolet irradiation were employed as the post-crosslinking treatment to prepare theelectrospun keratin/PEO (90/10) nanofiber membrane with good water tolerant. A comparative study between the performancesof the nanofiber prepared by different cross-linking methods were carried out. The results showed the three postcrosslinkingmethods were all effective for transforming the water-soluble keratin/PEO nanofiber membranes to the waterinsoluble membranes. However, the mechanical property, water sorption and swelling rate, molecular structure, thermalproperty, and crystallinity of the nanofiber membranes were all different due to the different post-crosslinking methods. Afterheat treatment, the tensile strength of the nanofiber membrane can reach 2.29 MPa, while that of the oxygen crosslinking andultraviolet crosslinking are 0.99 and 1.68 MPa, respectively. The water absorption and swelling of the oxygen and ultravioletcrosslinking membranes are relatively large, and the swelling rate is about 21%. The oxygen crosslinking membrane possessedhigher hydrophilicity and the contact angle is only 21°. In vitro cell culture studies showed that the post-crosslinkednanofiber membrane all presented excellent biocompatibility and supported cell adhesion and proliferation. The three ecofriendlylow-cost post-crosslinking treatments are all reliable and safe to prepare keratin/PEO nanofiber membrane with goodwater tolerant, which is potential for tissue engineering and biomedical applications.