A new rapid and reproducible method using microemulsion electrokinetic chromatography (MEEKC) combining field amplified sample injection and electroosmotic flow suppressant for the analysis of five quinolizidine alkaloids is developed in this paper. For the separation of five quinolizidine alkaloids, a running buffer composed of 1.2% (v/v) 1-butanol, 0.6% (v/v) ethyl acetate and 98.2% (v/v) 1mM Na 2 B 4 O 7 –2mM NaH 2 PO 4 buffer solution containing 21mM sodium cholate (SC) (pH 6.5) was developed. The resolution of the analytes was improved significantly by adding a divalent cation (e.g., Mg 2+ ) to the running buffer as an electroosmotic flow modification. In order to analyze trace quinolizidine alkaloids in traditional Chinese herbal medicines, field amplified sample injection (FASI) was applied to increase the detection sensitivity. The detection limits (defined as S/N=3) for the analytes could be as low as 0.0001μg/mL. This method was applied for the determination of quinolizidine alkaloids in real samples with simple extraction procedures, and the assay results were satisfactory.