The invention provides a tobacco seed primer, a preparation method of the tobacco seed primer, and a tobacco seed priming method, and relates to the technical field of plant cultivation. The tobacco seed primer provided herein is made from calcium chloride, gibberellin, betaine, proline, salicylic acid, sodium nitroprusside, and trehalose. The tobacco seed primer is capable of enhancing the resistance of tobacco seeds to low temperature stress, drought stress and low temperature and drought cross-stress, and is effective for increasing emergence rate of tobacco seeds and the quality of seedlings. In addition, the invention provides the preparation method of the tobacco seed primer; by dissolving calcium chloride, gibberellin, proline, salicylic acid, trehalose, betaine, and sodium nitroprusside sequentially in water, it is possible to effectively ensure the priming effect of the primer. In addition, according to the priming method of tobacco seeds provided herein, by using the tobacco seed primer provided herein, it is possible to enhance the cold resistance and drought resistance of tobacco seeds.