The invention relates to a storing and breeding method for preventing a tobacco germplasm resource from being lost, which is a storing and breeding method for preventing a tobacco germplasm resource from being lost due to vitality loss of seeds and belongs to the technical field of agricultural breeding upgrade. The method comprises the following steps of: (1) taking out tobacco seeds during different storage times in a germplasm bank at regular intervals, seedling in an artificial climatic chamber by group and calculating the germination percentage of the 14th day; and (2) continuously warehousing and storing the tobacco seeds with the germination percentage of 50 percent obtained in detection and counting, breeding the tobacco seeds the germination percentage of which is reduced to 30-50 percent in time and warehousing and storing. With the method, the vitality trend of the seeds in the germplasm bank can be known in time, any pollution on the environment cannot be caused and the storing cost can be reduced by reasonable germination percentage; in addition, the invention ensures the long-term safety storage of the tobacco germplasm resources and can prevent the germplasm resource from being lost caused by the infertility of the seeds due to the vitality loss of the seeds.