The research of chiral separation technology is of great significance for understanding the origin of life and promoting the application of chiral molecules. Herein, anionic chiral pillar[6]arene and cationic pillar[6]arene were designed and synthesized, and a chiral pillar[6]arene membrane was constructed by layer-by-layer assembly through electrostatic interactions. The transport rates of l-Ala and d-Ala in this channel were 14.33 and 1.86 μM cm-2 h-1, respectively, and the transport rate of l-Ala was 7.7 times that of d-Ala. In the transport experiment of Ala racemate, the ee value of l-Ala in the permeate after a single separation was 62%, indicating that this channel has a certain chiral separation ability. The effects of the assembly method, the number of chiral layers, and the driving force on the separation effect were further studied, and the selective transport mechanism was explored through the host-guest interaction at the molecular level and theoretical simulation. This strategy provides a new template for expanding the application of chiral pillar[n]arenes in the field of membrane separation.