In this research, a novel on-line two-step stacking preconcentration method by sweeping and micelle to solvent stacking in capillary zone electrophoresis was developed and validated for the simultaneous determination of quinolizidine alkaloids (sophocarpine, matrine and oxymatrine) in traditional Chinese medicines. Strychnine was used as an internal standard. The main parameters that affect the separation and sensitivity were investigated and optimized. Under the optimum conditions, the sensitivity enhancement factors obtained by the developed method for the analytes were from 42- to 52-fold. The method showed a good linearity over the range of 0.1–10.0 μg mL⁻¹ for sophocarpine, matrine and oxymatrine with the correlation coefficients (r) varying from 0.9992 to 0.9996. The limits of detection (S/N = 3) were 0.02–0.03 μg mL⁻¹. The intra-day (n = 8) and inter-day (n = 5) precisions of the method expressed as the relative standard deviation (RSD) were found to be less than 10%. The recoveries of the analytes by the method for the analysis of traditional Chinese medicines were in the range from 87.5% to 109.0% with RSDs (n = 3) less than 8.9%.