A reversed-phase HPLC method is described for determination of 5-fluorouracil in human serum. After being extracted with ethyl acetate 5-fluorouracil was analyzed by reversed-phase HPLC column (Zorbax ODS) and UV-detector (273nm) with tegaful as internal standard. The mobile phase was a mixture of methyl alcohol and water (20:80, V:V) at a flow rate of 1mL/min. The linear range was 0.04-50mg/L (r=0.9990) and the detectable limit was 10ng/mL. The average recovery was 96.85% and the coefficients of variation were 4.38% and 5.37% for within-day and day-to-day tests respectively.