A stacking system based on moving reaction boundary (MRB) for stacking and quantitative determination of oxymatrine (OMT) in urine samples was developed. The experimental conditions were optimized for the stacking of OMT as well as its separation. The optimized conditions were 20 mmol/L HCOONa (pH 10.70 adjusted by weak alkali of ammonia rather than strong alkali of sodium hydroxide) as sample buffer, 40 mmol/L HCOOH-HCOONa (pH 2.60) as stacking buffer, 100 mmol/L HCOOH-HCOONa (pH 4.80) as separation buffer, 1.4 kPa (3 min) sample phase injection and 1.4 kPa (7 min) stacking phase injection, 210 nm of detection wavelength, 21 kV of voltage. The linear response of OMT concentration ranged from 2.2 to 65 mg/L with high correlation coefficient (r = 0. 9991), the limit of detection (LOD) for OMT was 0.74 mg/L, and sensitivity was enhanced by 70 times. This method can be well used for quantification of OMT in urine samples with high sensitivity and can be further applied in the investigation of pharmacokinetics.