Ordered lamellar hybrid membranes are of great interest, due to their application as functional materials, including molecular sieving, biosensors, optical devices, and drug, as well as gene deliveries, but their fabrication remains challenging because three critical issues regarding synthesis of regular nanosheets, interfacing, and their spatial distribution in a polymer matrix are difficult to address simultaneously. Here, we fabricated a well-defined hybrid lamellar membrane through one-pot self-assembly, whose structure consisted of alternating layers of hydrophilic hyperbranched poly(ether amine) (hPEA) and polyhedral oligomeric silsesquioxane (POSS). The POSS-capped photosensitive hPEA initially formed ultrathin sandwich-like hybrid nanosheets (HNS) with a thickness of 5 ± 0.5 nm in an aqueous solution through a living crystallization-driven self-assembly, which subsequently, generated a highly ordered lamellar hybrid membrane with a uniform layer of spacing through direct self-stacking by filtration. The resulting membrane acquired a well-defined lamellar structure, thereby providing an ideal model to study molecular diffusion in confined spaces. Also, it could be used for the separation of molecules of different sizes or charges in water solution and is capable of being efficiently, effectively, and economically (known as 3E) applied for the purification of products of methyl orange and fluorescein from their precursor mixtures. This one-pot assembly of the hybrid polymer provides a facile and essential alternative to fabricate highly ordered hybrid membranes.