An analytical method was established for the determination of free glycerol in biodiesel by using liquid-liquid extraction technique and high performance liquid chromatography（HPLC） with evaporative light-scattering detector（ELSD）.The biodiesel sample was extracted with acetonitrile-water（75∶25）,followed by centrifugation.The obtained aqueous layer was analyzed by HPLC-ELSD.The testing conditions of HPLC-ELSD were optimized and the optimal conditions were as follows:separation column:Inertsil NH2 column（250 mm×4.6 mm,5 μm）,mobile phase:mixture of acetonitrile and water（75∶25）,flow rate:1.0 mL/min,injection volume:30 μL,drift tube temperature:28 ℃,gas pressure of nebulizer:360 kPa.The result indicated that,under the optimal conditions,the calibration curve was linear over the content of free glycerol in biodiesel in the range of 3.73-302.0 mg/kg with a correlation coefficient of 0.999 8,and the limits of detection（S/N≥3）were 2.1 mg/kg.The spiked recoveries were in the range of 98%-101% with relative standard deviations（RSDs） of 0.59%-1.8%.The method was sensitive,accurate and reproducible,and could provide an efficient way for the determination of the content of free glycerol in biodiesel.