Abstract: Antiviral substances of walnut (Juglans regia L.) leaves were extracted by different solvents and methods, and their antiviral activities were determined. The results showed that antiviral activity is dissimilar based on extracts from walnut leaves with different solvent and methods.For cold-leached extract with 95% ethanol, when concentration inoculated is 10 g/L, inhibition rate on tobacco mosaic virus(TMV) of the extract was the best, 100%; optimum extraction condition of this extract is: 95% ethanol as solvent, ratio of material to solvent 1:10, soaking time 18 h, extraction rate 21.42%. The inhibition rate of the extract on TMV infecting Nicotiana glutinosa is 100% when concentration inoculated is 5 g/L. Besides,an antiviral flavonoid A was isolated from ethyl acetate extract through column chromatography, thin layer chromatography and recrystallization. When its concentration inoculated is 0.5 g/L, inhibition rate of this compound on TMV infecting N. glutinosa is 85.55%. The result indicated that compound A is one of the main antiviral components in ethyl acetate extract.