To investigate chemical constituents from the aerial part of Ruta graveolens L. and their anti-Epstein-Barr virus (EBV) activity.The compounds from the dichloromethane fraction of Ruta graveolens L. were separated by column chromatography such as silica gel,MCI gel CHP-20,ODS,and Sephadex LH-20 and purified by preparative HPLC method,which led to the isolation of 30 compounds,including arborinine (1),2-hydroxy-3-isopropoxy-1,4-dimethoxy-10-methylacridanone (2) rutacridone (3),1-hydroxy-3-methoxy-N-methylacridone (4),20-Hydroxyrutacridon-epoxide (5),rutalinium (6),(-)-ribalinidine (7),ribalinium(8),(+)-ribaline (9),γ-fagarine (10),kokusaginine (11),haplopine (12),skimmianine (13),(-)-chalepin (14),chalepensin (15),(+)-rutaretin (16),xanthotoxin (17),psoralen (18),bergapten (19),7-preniloxicumarin (20),isoscopoletin (21),6-hydroxycoumarin (22),scopolin (23),graveoline (24),graveolinine (25),3,4,5-trimethoxycinnamyl alcohol (26),moskachan D (27),piperonyl acetone (28),rutin (29),1,3-disinapoyl-gentiobiose (30).The structures of all the isolated compounds were elucidated by spectroscopic methods (MS,1H NMR,13C NMR) and comparison with reported literatures.Compounds 2,7,9,12,20,22,23,26,27,28,30 were obtained from this plant for the first time.The isolated compounds 1-30 were evaluated for their anti-EBV activity.The results showed that six compounds inhibited more than 50% cleavage and replication of EB virus with concentrations of 30 μmol/L.Compounds 5 and 24 showed better anti-EBV activity with IC50 of 2.4 μmol/L and 4.8 μmol/L,respectively,which was higher than (+)-rutamarin in positive control group (IC50 = 7.0 μmol/L).