Abstract: An affinity capillary electrophoresis(ACE)method with bovine serum albumin(BSA)as chiral selector was developed to test the enantiomeric impurity in esomeprazole. The mechanism of enantioseparation of esomeprazole and its enantiomer via BSA was investigated by fluorescence and circular dichroism spectrum for thermodynamic parameters, enthalpy entropy-driven process and the conformational changes of BSA. The optimal separation condition obtained was a running buffer consisting of 20 mmol/L sodium dihydrogen phosphate and disodium hydrogen phosphate with 250 μmol/L BSA and 7% n-propanol at pH 8. 0, a positive voltage of 30 kV, separation temperature at 25 ℃ and detection wavelength at 302 nm. The resolution of esomeprazole and its enantiomer was more than 1. 8. The LOD and LOQ were 0. 8 μg/mL and 2. 0 μg/mL respectively, for the enantiomer of esomeprazole, and the linear range was 2-20 μg/mL. The average recovery of R-omeprazole was 100. 4%(RSD< 2. 0%). This method could be applied to test the enantiomeric impurity of esomeprazole drug substances.