The present invention belongs to the technical field of tobacco cultivation, particularly relates to a tobacco seed germination seedling strengthening agent, and further discloses a preparation method and a use method of the tobacco seed germination seedling strengthening agent. According to the present invention, the formula of the tobacco seed germination seedling strengthening agent is unique, quick lime, Bacillus subtilis, plant ash and a ginkgo extract are adopted as raw materials, and under the premise of no change of the existing seedling culture technical measures, the physiological effects of cold resistance, stress resistance and growth development promoting of seeds can be improved.