Abstract: In order to establish an efficient and accurate method for identification of resistance to tobacco black shank, experiments were performed using 4 tobacco varieties with different resistance characteristics. The toxin was added to the medium to study the effects of different concentrations of toxin stress and different stress time on tobacco seed germination and root growth. The results showed that the toxin stress of black shank disease could inhibit the germination and root growth of tobacco seedlings, and the inhibitory effect increased with the increase of toxin concentration. Different varieties have different tolerance to toxin stress. When the toxin was used at a concentration of 50% for 15 days, the ratio of the number of plants in the root growth medium of G28, YN116, Jingyehuang and Hongda was 79.38%, 70.74%, 33.55% and 13.36%, respectively. It was preliminarily determined that the optimal crude toxin concentration for identifying the resistance level of different varieties (germplasm) to tobacco black shank was 50% (protein content 13.53 μg/mL, sugar content 19.64 mg/mL), and the optimal stress time was 15 days. This method can effectively and accurately identify the resistance level of different varieties (germplasm) to tobacco black shank in a short period of time, and accelerate the breeding process of tobacco resistance to black shank.