The invention discloses a tomato seedling substrate and a preparation method thereof. The preparation method comprises the following steps: with cow dung dry-wet separation liquor and rice straw as base raw materials, regulating a carbon nitrogen ratio to provide an environment suitable for microbial growth, fermenting and composting, sieving composted materials obtained through microbial treatment to obtain materials with grain size of 1-5mm, sufficiently mixing, and then adding expansibility vermiculite and garden soil with certain fineness to improve the physical character and buffer property of a substrate so as to form the tomato seedling substrate, wherein the tomato seedling substrate comprises the following components in percentage by volume: 60%-80% of straw composted particles prepared from cow dung dry-wet separation liquor and rice straw, 10%-20% of expansibility vermiculite and 10%-20% of garden soil. The method is simple. The raw materials are abundant and cheap, environment pollution is reduced, nutrient is sufficient, the character is stable, the operation of topdressing during the seedling period of tomatoes can be completely or partially avoided, the seedling quality is improved, the pesticide application is reduced, the seedling quality is guaranteed, the labor and the cost are saved, and reliable guarantee is provided for the survival, the high-quality and high-efficient growth and the yield and quality of transplanted tomato seedlings.