Heterogenous and gradient structures are common in biological materials and essential for achieving exceptional mechanical properties from cost-effective components. Integrating glass flakes into this design concept holds great potential for enhancing the performance of bioinspired transparent materials, particularly through a synthesis approach that allows for precise control over their structural properties. In this study, we demonstrate that glass flakes modified with silane agents can spontaneously form a surface layer at the air–liquid interface. This interfacial assembly enables the layer-by-layer embedding of highly aligned glass flakes within the polymer matrix. By varying the aspect ratios of the glass flakes during film construction, we can create a controllable gradient, nacre-like architecture that offers an enhanced balance of strength and toughness, while maintaining transparency and haze comparable to the homogeneous structures. Lamination of both homogeneous and heterogeneous composite films further enables the evaluation of bending properties. The heterogeneous structure results in a superior combination of flexural strength, bending energy, fracture toughness, and work of fracture. Finite element simulations highlight the critical role of gradient structures and repeated sequences in redistributing stress and mitigating crack propagation. The interfacial assembly of glass flakes offers a versatile platform for optimizing the performance of bioinspired transparent materials by enabling precise and flexible manipulation of microstructures.