Electrospun keratin nanofiber fabricated with non-toxic solvents and crosslinking reagents are promising scaffolds for tissue engineering applications. Keratin from chicken feathers was solubilized in aqueous alkaline conditions and electrospun using aqueous poly(vinyl alcohol)(PVA)/citric acid solution as electrospinning polymer aid. Keratin/PVA nanofibers were successfully prepared containing 10, 20 and 30% keratin to PVA mass ratio. The increase in keratin concentration from 0 to 20% resulted in decreased viscosity of keratin/PVA solutions leading to a reduction in the fiber diameter from 565 nm in to 274 nm. Further decrease in viscosity with 30% keratin resulted in beads-on-fiber type of morphology. Nanofibers were thermally cross-linked through esterification reaction between carbonyl groups in citric acid and hydroxyl groups in PVA. Cross-linked nanofibers maintained their morphology and pore structure after soaking in water. The incorporation of keratin in PVA nanofiber was confirmed by FTIR and XPS results. Proliferation of fibroblasts after 14 days was higher in scaffolds containing 20% keratin which was attributed to the superior biological properties of keratin and higher surface area to volume ratio compared to 30% keratin/PVA scaffolds. Safer extraction, electrospinning and crosslinking conditions of keratin/PVA nanofiber are important for potential uses in tissue engineering.