OBJECTIVE: To establish a method for the concentration determination of vitamin A (VitA) in human serum, and apply it in healthy volunteers and hepatocirrhosis patients. METHODS: After liquid-liquid extraction, serum sample was determined by HPLC. Using VitA acetic ester as internal standard, the separation was performed on Kromasil C18 column with mobile phase consisted of methanol-water (98 ∶ 2,V/V) at the flow rate of 1 ml/min. The wavelength was set at 325 nm, and the column temperature was room temperature. The sample size was 20 μl. RESULTS: The linear range of VitA were 0.012 4-3.210 μg/ml(r=0.997 2,n=5), and the limit of quantification was 0.012 4 μg/ml. Inter-day and intra-day RSD ranged 1.66%-2.97%; sample recoveries were 98.18%-99.56%; extraction recoveries were 89.59%-91.38%. Average blood concentration of VitA were(0.71±0.08)μg/ml in 24 healthy volunteers and (0.28±0.06)μg/ml in 24 hepatocirrhosis patients. There was statistical significance in average blood concentration of VitA between healthy volunteers and hepatocirrhosis patients in different age groups (P<0.05). CONCLUSIONS: The method is simple, rapid, sensitive and accurate, and can be used for the concentration determination of VitA in serum of healthy volunteers and hepatocirrhosis patients.