OBJECTIVE： To establish the method for the determination of 25-hydroxyvitamin D [25（OH）D] in human plasma， and apply it in the clinic. METHODS： After liquid-liquid extraction， UPLC-MS/MS method was used to determine plasma sample. Using deuteron-25（OH）D3 as internal standard， the determination was performed on Zorbax Eclipse Plus C18 column with mobile phase consisted of water （containing 0.1％ formic acid）-methanol （containing 0.1％ formic acid） in gradient mode at flow rate of 0.35 ml/min， and the column temperature was 30 ℃. By ESI， positive ion scanning was conducted in MRM mode； the monitoring transition ion-pair was m/z 413.2→395.2 for 25（OH）D2， m/z 401.2→383.2 for 25（OH）D3 and m/z 404.3→368.2 for internal standard. RESULTS： The linear range of 25（OH）D2 and 25（OH）D3 were both 2-80 ng/ml （r＝0.998 0 and 0.999 9， n＝3）； the limit of quantitation was 2 ng/ml. RSDs of inter-day and intra-day were 0.78％-9.42％， and relative deviation were －2.90％-1.95％， respectively. Average plasma concentration of 25（OH）D in 78 health volunteers and 98 tuberculosis patients were（21.53±6.33）and（8.84±4.05）ng/ml， with statistical significance （P＜0.01）. CONCLUSIONS： The method is accurate， reliable and sensitive. It is suitable for quantitative analysis and pharmacokinetic study of 25（OH）D in human plasma.