Chameleons can rapidly modify their coloration by manipulating the arrangement of iridophores encompassing guanine crystals in their dermis, which enables incident light to undergo birefringence. Various biomaterials have been designed to mimic the arrangement of guanine crystals, yet instances of synthesizing bottom-up structures with anisotropy for optical functionality remain rare. In this study, we report a novel two-step gas diffusion method for in-situ synthesis of CaCO3 (Vaterite) within a hydrogel to fabricate a bio-photonic film displaying birefringence interference colors. This strategy enables the successful fabrication of an inorganic–organic photonic film with superior mechanical properties and flexibility, resulting in high transparency, superior toughness, and adjustable optical anisotropy. Through a combination of experiment and simulation results, we elucidated the critical role played by shape factor (distribution) and structural factor (particle size) in producing transmitted interference colors. Finally, the birefringent photonic films prepared using the dot-plate method can function as optical patterns accurately representing the “on”/“off” state within a binary system. This system offers high spatial resolution, excellent repeatability, and precise controllability of optical properties, making it applicable in fields such as information encryption and decrytion. Our study has successfully synthesized photonic material with anisotropic structure though in-situ method while elucidating the underlying mechanism governing their ability to generate transmission interference colors, thus opening up new avenues for transparent flexible polarized optics, imperceptible wearable devices, and advanced information encryption.