This study developed a simple and sensitive liquid chromatography method to quantify the multiple active alkaloids (matrine, sophocarpine, and sophoridine) from sophora flavescens ait in urine samples simultaneously. The results showed that liquid-liquid extraction using water as the solvent could effectively isolate all the analyses with the lowest matrix effects. A low concentration of diethylamine (0.07%, v/v) in the mobile phase improved dramatically the resolution of all the alkaloids with less interference from the matrix. Good linearity ranging from 0.025 to $1250.0;{mu}g/mL$ for all analytes was obtained, and the accuracy and precision varied from 73.25 to 87.86% and 1.4 to 2.2%, respectively. Finally, the suitability of the proposed method was demonstrated in the urinary drug excretion of 4 volunteers receiving an oral dose of a sophora flavescens ait extract. The intra and inter RSD were $leq$ 1.9%.