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Biomimetic protein-based platforms, with their hierarchical networks and optimal mechanical properties, show promising potential for hard tissue regeneration, including dental enamel. However, achieving aligned enamel-like apatite nanocrystals from organic matrices remains challenging. A simple organic-based approach to re-create the hierarchical enamel structure using water-based keratin films is reported. These films assemble via disulfide bridging into a fibrous organic network and birefringent spherulitic construction of predominant ordered β-sheet conformation. The flexible structure of keratin templates facilitates rearrangement of the secondary structures into α-helices upon mineralization, guiding the ordered growth of apatite nanocrystals. This system has shown potential in repairing early defective dental enamel lesions, restoring both optical appearance and mechanical properties. This study offers a promising, simple, and clinically-friendly method for developing novel protein-based matrices for hard tissue regeneration from naturally abundant sources.