A method based on in situ formed ionic liquids microextraction-ultra performance liquid chromatography(ISFILM-UPLC) was established for the determination of vancomycin in human serum.The samples were pretreated by vortex and centrifugation.[C6MIM] [Br] and NaPF6 were applied as the extracting agent,and Metronidazole as the internal standard.Methanol was applied to redissolve the precipitate.The separation was carried out on a BEH C18 column,and the mobile phase consisted of methanol and 0.05 mol/L monopotassium phosphate solution.The flow rate was 0.2 mL/min,the injection volume was 5 μL,and the column temperature was 18 ℃.The key parameters of this method such as the specificity,accuracy and precision were validated.This method showed good correlation with the immunoassay.It shows great potential for the application in clinical practice.