A method was developed for the separation and determination of 12 chiral polychlorinated biphenyls(PCBs),i.e.CB 45,CB 84,CB 91,CB 95,CB 131，CB 132,CB 136,CB 149,CB 171,CB 174,CB 176 and CB 183,in biological samples.The target chemicals were Soxhlet extracted ,cleaned up by gel permeation chromatography and multi-layer silica alumina column,and were further fractioned with a Florisil column.The chiral PCBs were identified and quantified by gas chromatography–mass spectrometry with electron impact ion source(GC-EI/MS).The peak resolutions of these chiral congeners ranged from 0.5 to 2.1.The relative standard deviations of the calculated enantiomeric fractions(EFs) in triplicates were less than 2%.The enantiomers of CBs 84,CB 91 and CB 174 in the standard solutions were separated by two different chiral columns.There was a slight discrepancy in the calculated EFs between the two columns,with relative standard deviations of 1.2%,0.2% and 0.2% for CB 84,CB 91 and CB 174,respectively,suggesting the high degree of accuracy and efficiency of the established method.