A novel flue-cured tobacco moisture seedling substrate comprises 40% carbonized rice husks, 30% fermented rice husks and 30% loess by volume. A preparation method of the substrate comprises the following steps: (1) adding water and uniformly stirring to prepare a material to be fermented after the rice husks are crushed; (2) uniformly mixing according to the weight ratio of the material to be fermented to urea of 50: 1; (3) mixing according to the weight ratio of the material to be fermented, an HM bacterial decomposition agent, rape seed cakes and rice bran of 1000: 0.2: 15: 20 to fully ferment so as to prepare fermented rice husks; (4) preparing the seedling substrate from 40% carbonized rice husks, 30% fermented rice husks and 30% loess by volume. The invention aims to provide the novel flue-cured tobacco moisture seedling substrate of which shape is difficult to destroy, the pH value is appropriate, the porosity is better, the permeability, the water retain and the nutrient-preserving capabilities are better, as well as the preparation method of the substrate.