Abstract: An HPLC method was developed for analysis of fingerprint of Kubu compound microemulsion formulation and determination of its main contents of matrine and oxymatrine. Samples were dissolved with 0.5% sulfuric acid after evaporated completely on water bath. Then, it was filtrated and adjusted to pH 10 to 11 with ammonia solution. Chloroform was then added and evaporate it completely after liquid-liquid separation. Ethanol was added to dissolve the residue. The microemulsion samples was thus prepared and separated with a Reprosil-par120-NH\-2 column, with column temperature of 30℃. The mobile phase was acetonitrile-ethanol-3% phosphoric acid solution (87:7:6). The similarity evaluation system for chromatographic fingerprint of TCM (2004) was employed on 10 samples and the median method was used to generate compound microemulsion reference fingerprint. The chromatographic peak is figured out by compared with reference substance. The content of matrine and oxymatrine in compound microemulsion was determinated by external standard method. The results showed that the similarity of fingerprint of 10 batches of microemulsion products were greater than 0.90. There were 7 common peaks in chromatographic, of which two peaks are for matrine and oxymatrine respectively. The linear relationship of the matrine in the range of 3.63-58.0 μg/mL (r=0.999 1) and the oxymatrine in the range of 4.50-72.0 μg/mL (r=0.999 0) are good. The average recovery of matrine was from 98% to 99%(RSD between 0.77%-2.6%, n=9)and the average recovery of oxymatrine was from 96% to 99% (RSD between 1.0%-2.0%, n=9) . The limit of detection (LOD) were 0.20 mg/L and 0.23 mg/L respectively, and the limit of quantification (LOQ) were 0.67 mg/L and 0.75 mg/L respectively for matrine and oxymatrine; The average content of matrine and oxymatrine were 57.07, 43.55 mg/L in 10 batches of microemulsion samples, respectively. The HPLC fingerprint was informative and characteristic, and has good specificity to control the overall quality of Kubu compound microemulsion formulation. The HPLC method has high accuracy, repeatability and simple operation. Combined with HPLC fingerprint, the overall quality of Kubu compound microemulsion formulation can be controlled comprehensively and effectively.