A new method of electrostacking-sweeping micellar electrokinetic chromatography was developed for the simultaneous determination of two alkaloids,matrine and sophoridine,in sophora flavescens.The determination conditions including pH value,concentration of NaH2PO4 and CTAB,injection time and voltage were optimized.A best separation of two alkaloids was obtained on an uncoated fused silica capillary column(60 cm×50 μm,effective length 47 cm) by using a buffer of 20 mmol/L NaH2PO4-0.8 mmol/L CTAB-100 mmol/L Tris（containing 10% isopropanol） at pH 8.9 and a constant temperature of 25 ℃.The UV detection wavelength was set at 211 nm.The applied voltage,separation voltage and injection time were -10 kV,-20 kV and 480 s,respectively.Under the optimal conditions,the calibration curves were linear in the range of 46-736 μg/L for matrine and 50-792 μg/L for sophoridine with their detection limits(S/N=3) of 2.41 μg/L and 1.11 μg/L,respectively.The spiked recoveries were in the ranges of 93%-109% and 89%-103%,respectively,with RSDs less than 5%.Compared with the sweeping-MEKC method, the electrostacking-sweeping-MEKC method raised the sensitivity by two orders of magnitude.The method showed high separation efficiency and good sensitivity,and was applied in the simultaneous separation and determination of two alkaloids in sophora flavescens with satisfactory results.