OBJECTIVE： To establish the method for the determination of doxofylline in human serum. METHODS： Online two-dimensional column switching-HPLC was adopted to determine the concentration of doxofylline in human serum. First- dimensional chromatographic column was Waters C18 column， and middle column was SC2. First-dimensional and second- dimensional column mobile phrase were methanol-water （70 ∶ 30， V/V）. The detection wavelength was 273 nm， and column temperature was 40 ℃. Sample volume was 10 μL. RESULTS： The linear range of doxofylline were 0.5-50.00 μg/mL （r＝0.999 9）， and the detection limit was 0.01 μg/mL. The quantitative limit was 0.5 μg/mL. RSDs of intra-day and inter-day were 1.51％-1.89％ and 1.52％-1.92％ （n＝5）. The accuracy were 97.91％-104.19％ （n＝5）. Extraction recoveries rate were 91.63％-93.44％（RSD＜2.00％，n＝3）. RSD of matrix effect were lower than or equal to 3.01％ （n＝6）， and RSD of stability test was lower than 5.00％ （n＝6）. After 3 patients were given intravenous injection of Doxofylline and glucose injection（0.3 g/d） up to steady state， the serum concentrations of doxofylline were 3.23， 3.35， 3.68 μg/mL before medication on the next day （RSD＝2.28％， 2.34％， 2.14％， n＝5）. CONCLUSIONS： The method is simple， rapid， accurate and suitable for the determination of plasma concentration of doxofylline.