Abstract: The chiral separation of nicotine enantiomersS-(-)-nicotine and R-(+)-nicotine is one of current research focuses and a key step on stereoselective pharmacokinetics study of nicotine. The research progress on chiral separation methods of nicotine enantiomers in recent 40 years was reviewed and discussed, including liquid chromatography, supercritical fluid chromatography, gas chromatography, capillary electrophoresis, nuclear magnetic resonance methods, etc. The development in this field was prospected, wherein liquid chromatography and supercritical fluid chromatography containing chiral stationary phases applied with modified polysaccharides and macrocyclic antibiotics were most widely used due to their advantages being fast, efficient, sensitive and environmentally friendly.