The invention discloses a rice tray-type seedling culture substrate and a preparation method thereof.The preparation method comprises: first, preparing carbonized straws, rice field dry soil, a compound fertilizer and a soil sterilizer according to a weight ratio, wherein the carbonized straws is 60% by weight, the rice field dry soil is 39% by weight, the compound fertilizer (a ratio of N, P and K is 15:15:15) is 0.05-0.5% by weight, and the balance is the soil sterilizer; second, mechanically crushing and mixing the carbonized straws, the rice field dry soil, the compound fertilizer, the soil fertilizer and a plant growth regulator to obtain the rice seedling culture substrate, and subpackaging the substrate according to the requirements of seedling factories.Compared with traditional straw compost, the prepared substrate enables high and thorough composing speed of the straws, has complete nutrition and is light in weight and good in quality; the weight of the substrate is less than 50% as heavy as the traditional substrate; the problem with nutrition and weight balance is solved, seedlings can grow healthily, seedling emergency after mechanical transplantation is high, and the seedlings turn green early.