A rapid, reproducible and high-sensitivity NACE-ESI-MS method was developed for the analysis of sophoridine, matrine, sophocarpine and oxymatrine in the roots of Sophora flavescens Ait. and S. tonkinensis Gagnep. Field-amplified sample stacking with electromigration-injection (FASS-EMI) was first used in NACE for the on-line concentration of the alkaloids. The conditions of NACE separation, FASS-EMI stacking and MS detection were systematically optimized. The optimum NACE buffer was an electrolyte containing 50 mM ammonium acetate, 0.5% acetic acid and 30% ACN in methanol. The sensitivity was improved by about 100-fold by the FASS-EMI technique, which was further improved by more than 1000-fold with MS detection. The RSDs (n = 6) of the relative migration time and relative peak area of each peak were less than 0.3 and 2.4% for intra-day and less than 5.1 and 6.0% for inter-day, respectively. The LODs (S/N = 3) of analytes were determined to be 0.0210-0.0446 ng/mL. A bioanalytical method based on this NACE-ESI-MS method may be developed for the analysis of the alkaloids in biological sample matrices (plasma, urine, etc.) after effective ion removal.