The invention provides a substrate for breeding tobacco seedlings in floating mode and a manufacture method thereof. The substrate comprises swelling perlite and vermiculite, and also comprises rotten peanut bran; and the mass percentage of various compositions in the substrate is 60 to 90 percent of rotten peanut bran, 4 to 19 percent of perlite, 4 to 19 percent of vermiculite, and the balance being impurities. The fermented rotten peanut bran is used to breed seedlings, thereby being sanitary and having no weed phenomenon; and the peanut bran, the perlite and the vermiculite are mixed according to certain proportion, and are reasonably sterilized by steam, thereby not only increasing the hydroscopic property and permeability of the peanut bran, but also balancing the weight of a floating plate and reducing bran leakage. Because the production method adopts uniform mixing after crushing to produce the substrate, the substrate is even and consistent and improves the consistency of the nutrition resistance and growth; besides, the production method has the advantages of low cost, land, labor and work conservation, high intensification degree, high seed utilization efficiency, convenient transplanting and transportation, and resource conservation, and gets rid of dependence on grass carbon.