Abstract: OBJECTIVE To develop a simple and sensitive HPLC with fluorescence detection method for simultaneous determination of hydroxytyrosol, salidroside and tyrosol in wine-fried Ligustri Lucidi Fructus. METHODS The analysis was performed on a Amethyst C₁₈-P column(250 mm×4.6 mm, 5 μm) with isometric elution using water and acetonitrile(92:8). The detection wavelengths were at 280 nm and 310 nm for excitation and emission. The column temperature was 35℃. RESULTS The good linearity was in the certain concentration range, and the limit of quantitation of hydroxytyrosol, salidroside and tyrosol were 23.0, 10.5, 9.82 ng·mL^-1, respectively. The range of content of hydroxytyrosol, salidroside and tyrosol in wine-fried Ligustri Lucidi Fructus were 642.9-867.2 μg·g^-1, 1 932.3-15 357.5 μg·g^-1, 239.1-638.1 μg·g^-1, respectively. CONCLUSION The developed HPLC-Fluorescence detection method is sensitive, selective and accurate for simultaneous determination of hydroxytyrosol, salidroside and tyrosol in wine-fried Ligustri Lucidi Fructus.