Abstract: Objective: A reversed-phase high performance liquid chromatography(HPLC) method was developed for determination of fluconazole concentration in human serum. Methods: A reversed phase HPLC method was developed after the serum samples were extracted by trichloroacetic acid solvent. HPLC conditions were set as follows: Column: Lichrospher-C18，250 mm 4. 6 mm，5 m; Mobile phase was acetonitrile: water: phosphoric acid(22∶ 78∶ 0. 28, adjusted to pH 4 with ethidene diamine) . Flow rote: 1. 0 ml /min, Detection wave length: 295 nm, the column temperature was 20 ℃. results: Assay linearity was obtained in the range of 0. 1-6 g /ml (r = 0. 999 9) ; the relative standard deviation between intra-day and inter-day was lower than 10%. The recovery rate of three different concentrations(low，middle and high concentration) was between 85. 93% and 100. 60%. Conclusions: The method is rapid, accurate and reproducible. It is suitable for therapeutic drug monitoring.