Abstract: A headspace solid phase microextraction（HS-SPME） followed by gas chromatography had been developed for the determination of ethanol in fermentation solution of xylose.Various parameters including the type of fibers,extraction time,extraction temperature,pH value,ionic strength and desorption conditions were investigated for the optimization of HS-SPME performance for ethanol in fermentation solution of xylose.The high purity nitrogen with a flow rate of 4.0 mL/min,and DB-ALC2 capillary column（30 m×0.53 mm,2 μm） were used for separation.The oven temperature was programmed at 40 ℃.Under the optimal conditions,the quantitative results showed that the linear range of ethanol was 50-300 μg/L by using tert-butyl alcohol as internal standard.The detection limit（3σ） was 4.19 μg/L.The relative standard deviations for the determination of studied compound was less than 6%,and the recovery range was ranged from 91.01% to 97.90%.The proposed method had been successfully applied to determine ethanol in fermentation solution of xylose with satisfactory results.