The extraction and determination of cytisine, sophocarpine, matrine, sophoridine and oxymatrine in Sophora flavescens Ait. were performed using subcritical water extraction and capillary electrophoresis with field-amplified sample stacking. The effects of extraction temperature, pressure, time and cycle number on the extraction yields were investigated systematically for accelerated solvent extraction with ethanol (ASE) and accelerated solvent extraction with water (subcritical water extraction, SWE). The extraction yields obtained using SWE, ASE, water ultrasonic extraction and chloroform soaking extraction methods were compared. The electrophoresis separation buffer was monosodium phosphate (pH 3.0; 110mM)-isopropanol (85:15, v/v). The effect of phosphoric acid added to the sample matrix on the reproducibility of the peak heights of the analytes was also examined. Cytisine, sophoridine and oxymatrine showed good linearity (R²>0.999) within 0.125–4.0μg/mL, and sophocarpine and matrine exhibited good linearity (R²>0.998) within 0.0625–2.0μg/mL, with the detection limits in the range of 0.004–0.0013μg/mL. The five alkaloid contents in medicinal plants from different sources and Sophora instant granule were determined and compared.