Abstract: To establish a molecular detection method of new target for tobacco black root rot caused by Thielaviopsis basicola, β-tubulin gene was selected as a new target and a pair of species-specific primers Tbas-tub2F/Tbas-tubR was designed to detect T. basicola by PCR. The specificity, sensitivity and early diagnosis technology of the primers were assayed. The results showed that an expected 254 bp band could be amplified only from T. basicola and no PCR product was amplified from any of the other nineteen fungi and oomycetes and negative control, which suggested that the primers could be used to detect T. basicola specifically. The detection sensitivity of Tbas-tub2F/Tbas-tubR primers was 10 fg genomic DNA of T. basicola. The pathogen could be detected from tobacco tissues 24 hours post inoculation thus the primers were good for early accurate diagnosis of tobacco black root rot disease. In this study we developed an alternative new target for both molecular detection and early diagnosis of tobacco black root rot caused by T. basicola.