The invention discloses a method for improving freezing resistance of tobacco seedlings, and belongs to the field of agriculture. A water culture method is adopted, the freezing resistance of the tobacco seedlings is improved through three stages of low-temperature domestication, de-domestication and low-temperature treatment, and the stress resistance related gene expression quantity of the tobacco seedlings is improved when the tobacco seedlings are subjected to low temperature again through de-domestication for 12-24 hours, so that the redox and osmotic regulation balance of the plants are promoted to keep stable. The method solves the problem of insufficient freezing resistance of tobacco in cold regions at present, has important significance for stable development of tobacco production in cold and cold tobacco regions and continuous income increase of tobacco growers, and provides a certain basis and exploration approach for avoiding influence of short-time warming of overwinter crops on domestication effect and avoiding harm of short-time cooling of spring sowing crops through domestication.