OBJECTIVE： To establish a method for determination of fluconazole concentration in human plasma. METHODS： UPLC-MS/MS method was adopted to determine plasma after precipitated with acetonitrile. Using isotope fluconazole-d4 as internal standard， the determination was performed on ACQUITY UPLC BEH C18 column with mobile phase consisted of 0.1％ formic acid-acetonitrile （gradient elution） at the flow rate of 0.3 mL/min. The column temperature was 40 ℃， and the sample size was 3 μL. ESI was used for positive ion scanning by multiple reaction monitoring mode. The ion pairs for quantitative analysis were m/z 307.1→220.0 （fluconazole） and m/z 311.1→223.0 （internal standard）. RESULTS： The linear range of fluconazole was 10-5 000 ng/mL （r＝0.998 1）. The limits of quantitation was 10 ng/mL. RSDs of intra-day and inter-day were less than 8％； accuracy ranged 95.8％-106.7％. The extraction recovery ranged 97.3％-107.3％ （RSD＜5.0％， n＝6）， and matrix effect， dilution effect and residual effect didn’t influence quantitative analysis of the substance to be measured. CONCLUSIONS： The method is simple， rapid， specific and accurate， which can be used for therapeutic drug monitoring and pharmacokinetic study of fluconazole.