Abstract: To establish a system for determining the pathogenicity of tobacco fusarium root rot pathogen in seedling stage, improve the sensitivity and accuracy of identifying fusarium root rot resistance in tobacco varieties, the inoculation method, seedling age, temperature, pathogen content, and soil moisture content were screened and optimized respectively. In the inoculation system, the root-drenching method with spore suspension had high stability and the tobacco seedlings showed a relatively high degree of disease incidence. Tobacco seedlings at the seedling age of 40 days were susceptible to root rot. The disease index of root rot of tobacco seedlings in the inoculation system was the highest when the cultivation temperature was 28 °C. The degree of root rot was high when the soil water content was 40% and 80%. In this study, the established determination system for the pathogenicity of tobacco fusarium root rot is conducive to the occurrence of root rot and has good stability, which helps to more accurately identify its Fusarium root rot resistance in the early growth stage of tobacco.