A new, simple, and quick method for the simultaneous determination of three alkaloids (matrine, sophoridine, and oxymatrine) in composite preparations by nonaqueous capillary electrophoresis has been developed. A running buffer composed of 60% acetonitrile + 0.75% acetic acid + 25 mM ammonium acetate in methanol was found to be the most suitable for this separation. The limits of detection for three alkaloids were over the range of 0.45-0.6 μg/ml. In the tested concentration range, good linear relationships between peak areas and concentrations of the analytes were obtained. This method has been successfully applied to simultaneous determination of the three alkaloids with recoveries over the range of 95.6 to 97.2%. This new non-aqueous capillary electrophoresis method established has been successfully used for the simultaneous separation and determination of sophoridine, matrine and oxymatrine in complex preparations in 5 minutes. © 2022 Academic Publishing House. All rights reserved.