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The purpose of the present study was to establish a high-performance liquid chromatography method for the determination of the oxymatrine content of radix Sophorae tonkinensis and to comparatively analyze the content of oxymatrine in radix Sophorae tonkinensis from different origins. C18 was used as a chromatographic column (250 mm × 4.6 mm, 5 μm), methanol/ 3% phosphoric acid in water (80:20) as the mobile phase, 220 nm as the ultraviolet wavelength detection, 1 mL/min as the flow rate and 30°C as the column temperature. All samples were subjected to organic solvent extraction with ultrasonic treatment before the oxymatrine content from radix Sophorae tonkinensis was measured. Oxymatrine in the range of 30 μg/mL to 150 μg/mL had a good linear relationship (r=0.9998), and the average recovery was 99.22%, suggesting that this method is accurate and has high reproducibility. The established high-performance liquid chromatography method for determining oxymatrine content from radix Sophorae tonkinensis is efficient and stable. This may be an evaluation criteria used for quality control given that different origins of radix Sophorae tonkinensis have wide-ranging differences in oxymatrine content.