Precise separation of complex mixtures is of great significance. Membrane technology is attractive for advanced molecular separation due to its energy efficiency and environmental friendliness. Herein, charged membranes with β-cyclodextrin (CD) scaffolds are prepared for precise sieving of complex molecular mixtures. Interfacial polymerization between trimesoyl trichloride and CD-based zwitterions could generate a selective separation layer composed of the CD-scaffolded porous network and plentiful quaternary ammonium groups. Both hydrophilic and hydrophobic channels existing in the separation layer endow the membrane with high permeance of polar and nonpolar solvents. The sieving mechanism of the CD-based membrane is investigated, demonstrating that the Donnan effect and size exclusion have different influences on anionic, cationic and neutral molecules during filtration. The size- and charge-dependent molecular sieving is determined by the inner cavities of CDs, outer spaces between CDs and the quaternary ammonium groups of the cross-linked CD network. This charged CD-based membrane can stably and precisely discriminate molecules with different charges, molecular weights or shapes. The charged CD-based membrane can precisely discriminate molecules with different charges, molecular weights, or shapes, which was determined by the inner cavities of CDs, outer spaces between CDs, and quaternary ammonium groups of the CD network.