To validate the technique of molecular hybridization on the detection of Strawberry mottle virus (SMoV), total nucleic acid, which extracted from strawberry leaves using CTAB approach, was adopted as a template to be amplified with specific primers in non-coding region of SMoV. And then, sequences of SMoV were obtained after cloning and sequencing phases. The results of phylogenetic analysis indicated that there was a far relative between the isolate in this research and the reported isolates in GenBank, the isolate in the study formed a small unattached branch in phylogenetic tree. Using plasmid DNA with viral specific fragment as a template, a cDNA probe labeled with digoxigenin was prepared by PCR. SMoV could be efficiently detected by the cDNA probe in the test material of strawberry leaves infected by SMoV, as well as in the extreme case with 10 folds dilution solution of total nucleic acid. To our knowledge, this is the first report on the detection of SMoV by cDNA probe.