The antiviral substances of walnut leaves were extracted by different solvents and methods, and its antiviral activities were determined. The results showed that antiviral substance activity is dissimilar based on extracts from walnut leaves with different solvent and methods. Inhibition rate on TMV of the extract with 95% (V/V) ethanol is the best, 100%; Optimum condition of the cold soak are: 95% (V/V) ethanol is used as solvent, the ratio of material to solvent is 20/100 (1∶10), soak time is 18 h. The inhibition rate of the extract to TMV infecting Nicotiana glutinosa is 100% under optimum condition.