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).