To accurately test the content of virus in tobacco after treated by antivirus substance, a real-time PCR assay system was established. A bacteria strain antagonistic to the cucumber mosaic virus (CMV), B6, was isolated from a field of tobacco. The isolated and purified antivirus substance is referred to as B6p, its relative molecular weight is 40.6kDa. The virus multiplication characteristics in B6p treated host were analyzed with a round leaf method, and the results showed that B6p inhibited the multiplication of virus in the host, and the multiplication action weakened with the increase of antivirus substance concentration.