Abstract: This study was established a method of ultra performance liquid chromatography tandem fluorescence detector with liquid-liquid extraction for the rapid separation and determination of tyrosol and hydroxytyrosol in olive oil. The mobile phase was mixture with 0.2% (v/v)formic acid acetonitrile and 0.2% (v/v)formic acid water at a flow rate of 0.15 mL/min after extracted by ethanol. Using the BEH (1.0 mm i.d.×50 mm 1.7 μm)column. The fluorescence detector was detected at the excitation wavelength of 300 nm and the emission wavelength of 350 nm. The whole analysis process is only 10 min. The limit of detection (LOD)and limit of quantification (LOQ)of tyrosol and hydroxytyrosol were 0.11, 0.13 μg/g and 0.31, 0.39 μg/g, respectively. The recovery rate of the 2 components at the high, middle and low levels of 3 concentration levels was higher than 89.52%. The determination of tyrosol and hydroxytyrosol in 20 batches of olive oil from different areas were carried out under the optimal conditions. The contents of tyrosol and hydroxytyrosol were range from 1.28~20.34 μg/g and 1.03~21.22 μg/g, respectively. This method is simple, fast and reproducible, and could applicable for determination of tyrosol and hydroxytyrosol in olive oil.