Nanoparticles, which have unique properties, have attracted growing attention in enantiomeric separation nowadays. In this paper, an L-cysteine functionalized gold nanoparticle (L-Cys-GNP) based capillary column was prepared and applied in separating drug enantiomers in capillary electrochromatography (CEC) with lactobionic acid (LA) as a chiral selector. Compared with bare fused-silica capillary columns, the capillary columns modified with L-Cys-GNPs showed excellent chiral separation performance. A series of parameters affecting the enantiomeric separation were systematically investigated.