An application of on-line coupling of capillary electrophoresis and mass spectrometry (CE–MS) to the chiral separation of phenoxy acid herbicide enantiomers was investigated. As an ionization method, electrospray ionization (ESI) is used for a CE–MS interface. Generally, nonvolatile additives in separation solutions sometimes decrease the MS sensitivity and/or signal intensity. In this study, however, heptakis(2,3,6-tri- O-methyl)-β-cyclodextrin (TM-β-CD) was used as a chiral selector and it migrated directly into the ESI interface. By using the negative-ionization mode along with a methanol–water–formic acid solution as a sheath liquid and nitrogen as a sheath gas, stereoselective separation and detection of three phenoxy acid herbicide enantiomers were successfully achieved with a 20 m M TM-β-CD in a 50 m M ammonium acetate buffer (pH 4.6).