A quantitative analysis method for fudosteine in human serum by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI/MS/MS) was established, which shows high sensitivity and selectivity. The mobile phase composition was 75% 20 mM acetic acid and 25% acetonitril, which was pumped at a flow rate of 0.40 mL/min. The overall chromatographic run time was approximately 7 min. The autosampler was set with an injection volume of 10 μL. The calibration curve was linear in the concentration range of 0.1~15.0 µg/mL. The coefficient of determination (r) was greater than 0.9998. This method has been fully validated and shown to be specific, accurate and precise. The method was simple, rapid and the sample preparation was minimal. It was successfully applied to the analysis of healthy volunteer.