The invention discloses rice seedling nursing soil, as well as a preparation method and application thereof, and relates to the field of rice seedling nursing. The problems of straw combustion and difficulty in soil taking during rice seedling nursing in spring are solved. The rice seedling nursing soil is prepared from the following components in parts by weight: 100 to 200 parts of soil, 80 to 90 parts of straw powder, 10 to 20 parts of a compound microbial agent, 0.1 to 0.15 part of potassium dihydrogen phosphate, 0.1 to 0.4 part of ammonium sulfate and 0.1 to 0.3 part of quicklime, wherein the compound microbial agent is prepared from the following components: pseudomonas syringae, bacillus thuringiensis, bacillus subtilis, bacillus licheniformis, trichoderma and lactobacillus plantarum, and the mass ratio of the pseudomonas syringae, the bacillus thuringiensis, the bacillus subtilis, the bacillus licheniformis, the trichoderma and the lactobacillus plantarum is 1.5:1:1:1:1:1.5. According to the rice seedling nursing soil and the preparation method and application thereof, insect killing, sterilization, pest and disease damage prevention and control and increase of transmission of oxygen in the soil can be simultaneously implemented, growth and reproduction of microorganisms are facilitated, pollution is avoided, and the yield is high.