Au20 (PP3 )4 Cl4 (PP3 =tris(2-(diphenylphosphino)ethyl) phosphine), abbreviated as Au20 , is the only Au nanocluster with an intrinsically chiral core without a chiral environment (chiral ligands or Au-thiolate staples), making it a unique object to understand chiral evolution and explore chiral applications. Unfortunately, the synthesized Au20 is racemic, and its enantiomers have not yet been separated. Herein, we report a supramolecular assembly strategy with α-cyclodextrin (α-CD) to afford enantiopure Au20 in bulk, and an enantiomer excess (ee) value of as-separated Au20 of 97 %. As a result of its high purity, the distinctive optical activity of Au20 , which originates from electronic transitions confined in chiral cores, is fully explored. Theoretical studies reveals that the enantioseparation results from the preferential self-assembly of α-CD with organic ligands on the surface of right-handed Au20 .