Four strains were isolated from lettuce with leaf spot disease by tissue isolation method and identified as Stemphylium spp. with ITS sequences analysis. An attenuated strain with debilitated pathogenicity and slower growth rate named WS–01 was found by artifical inoculation. The cellulose CF11 method was used to extract dsRNA to determine the relation between the debilitated pathogenicity and its dsRNA mycovirus, three dsRNAs ranged from 1 kb to 5 kb were detected in the WS–01 strain. High temperature-ribavirin-hyphal tip isolation method for elimination of virus was conducted. The results showed that the pathogenicity of mycovirus-eliminated strain WS–01–D was enhanced and the phenotype returned to normal compared to the original strain, indicating the abnormal phenotype of strain WS–01 was related to its dsRNA.