The invention relates to a tobacco floating seedling culture medium and a preparation method thereof. The tobacco floating seedling culture medium contains 70-90 percent of decomposed peanut chaff and 10-30 percent of river sand. The preparation method comprises the steps of: removing impurities in peanut hulls, crushing the peanut hulls into peanut chaff with grain size being 3-6mm, regulating the water content of the obtained peanut chaff to 60-70 percent, adding HM decomposing agent and urea, evenly mixing the HM decomposing agent and the urea, conducting stack retting, sterilizing the decomposed peanut chaff and fully and evenly mixing the decomposed peanut chaff with the river sand to obtain the tobacco floating seedling culture medium, wherein at a decomposing high-temperature stage, the internal temperature of a stack is kept to be 55-65DEG C, the stack is stirred to decrease temperature when the internal temperature of the stack increases to more than 65DEG C, and the decomposed peanut chaff is obtained when the internal temperature of the stack is restored to normal temperature and does not increase. The tobacco floating seedling culture medium has the advantages that the grain size is moderate, the medium is loose and air-permeable and can form an aggregate structure, the air permeability and the water holding capacity are good, the physical and chemical properties are stable, the activities of tobacco leaf nitrate reductase and invertase can be obviously improved, the development of tobacco roots is promoted and the adverse rhizosphere effect does not exist.