Background : Development of new real-time PCR diagnosis method for simultaneous diagnosis of Cylindrocarpon destructans and Fusarium solani, causative fungi of ginseng root rot disease. C. destructans and F. solani are known to be the major pathogens of ginseng root rot disease. Root rot caused by these pathogens is a disease that is difficult to control because the disease progresses slowly and it is difficult to diagnose early and even when symptoms of plant seeding are present, the disease is already spread in the roots. Diagnostic methods to detect the presence or absence of ginseng roots rot fungi in soil before ginseng cultivation are currently being used as a method for controlling. However, commercialized soil extraction kits and PCR diagnostics have cost, diagnostic time, and single diagnostic problems, and need to develop new diagnostic methods. Methods and Results : Primers and probes in the beta-tubulin 2 gene were designed for species-specific detection. In silico analysis, the detection rate of C. destructans was 100% and the detection rate of F. solani was 95%. The multiplex real time PCR optimization conditions including the internal control were established. The analytical sensitivity using positive samples was 10 copies/㎕ for C. destructans and 10 copies/㎕ for F. solani. As a result of performance comparison test with conventional PCR diagnosis methods, it was confirmed that the developed multiplex real time PCR method has the same or better performance in terms of sensitivity. In the developed soil extraction kit, the extraction time was reduced and the extracted DNA quality was improved, compared to the used soil extraction kit. Conclusion : From the above results, we expect that the developed C. destructans / F. solani multiplex real time PCR diagnosis method and soil extraction kit will be useful for real-time monitoring of ginseng root rot pathogenic fungi in the soil of ginseng cultivation area and diagnosis of suitability of ginseng cultivation area.