Abstract: OBJECTIVE To investigate the antiviral activity and chemical composition of ethyl acetate extract of Radix isatidis. METHODS Each component of total ethyl acetate extracts was detected by solvent and chromatogram， the anti-HSV-1 activities of each component were detected by MTT assay in vitro， each component of total ethyl acetate extracts was analyzed by UPLC-Q-TOF-MS， and the data were analyzed by Markview and Excel software to find the difference among different components. According to statistics， the components related to antivirus activities were obtained. RESULTS According to the similarity of chemical composition， 16 components were extracted and separated from ethyl acetate extract of Radix isatidis by chromatography， and 8 components had antiviral activity. 49 chemical components were detected by UPLC-Q-TOF-MS from ethyl acetate extracts and their chemical structures were confirmed. Difference analysis of chemical components discovered 12 chemical components such as alkaloids， organic acids and amino acid derivatives associated with antiviral activity. CONCLUSION The ethyl acetate extract of Radix isatidis mainly contains alkaloids， organic acids， nucleosides， flavonoids， anthraquinones and amino acids， wherein the alkaloids， organic acids and amino acid derivatives are closely related to the antiviral activity.