The separation of chiral drugs continues to pose a significant challenge. However, in recent years, the emergence of membrane‐based chiral separation has shown promising effectiveness due to its environmentally friendly, energy‐efficient, and cost‐effective characteristics. In this study, we prepared chiral composite membrane via interfacial polymerization (IP), utilizing β‐cyclodextrin (β‐CD) and piperazine (PIP) as mixed monomers in the aqueous phase. The chiral separation process was facilitated by β‐CD, serving as a chiral selective agent. The resulting membrane were characterized using SEM, FT‐IR, and XPS. Subsequently, the chiral separation performance of the membrane for DL‐tryptophan (Trp) was investigated. Lastly, the water flux, dye rejection, and stability of the membrane were also examined. The results showed that the optimized chiral PIP0.5β‐CD0.5 membrane achieved an enantiomeric excess percentage (ee%) of 43.0% for D‐Trp, with a solute flux of 66.18 nmol·cm−2·h−1, and maintained a good chiral separation stability. Additionally, the membrane demonstrated positive performance in the selective separation of mixed dyes, allowing for steady operation over a long period of time. This study offers fresh insights into membrane‐based chiral separations.