Abstract Biomimetic reconstruction of tooth enamel is a significant topic of study in materials science and dentistry as a novel approach to the prevention, restoration, and treatment of defective enamel. We have developed a new amelogenin-containing chitosan hydrogel for enamel reconstruction that works through amelogenin supramolecular assembly, stabilizing Ca-P clusters and guiding their arrangement into linear chains. These amelogenin Ca-P composite chains further fuse with enamel crystals and eventually evolve into enamel-like co-aligned crystals, anchored to the natural enamel substrate through a cluster growth process. A dense interface between the newly grown layer and natural enamel was formed and the enamel-like layer improved the hardness and elastic modulus compared with etched enamel. We anticipate that this chitosan hydrogel will provide effective protection against secondary caries because of its pH-responsive and antimicrobial properties. Our studies introduce an amelogenin-containing chitosan hydrogel as a promising biomaterial for enamel repair and demonstrate the potential of applying protein-directed assembly to biomimetic reconstruction of complex biomaterials.