The invention relates to the technical field of side slope treatment and discloses a spray-seeding substrate for ecological reafforestation of high and steep side slopes, a preparation method for the spray-seeding substrate and an application of the spray-seeding substrate. The spray-seeding substrate contains the following raw materials in parts by weight: 330-420 parts of planting soil, 20-30 parts of peat soil, 1.5-5 parts of organic fertilizer, 1-3 parts of grass fibers, 1-3 parts of wood sawdust, 0.03-0.05 part of water retention agent, 0.02-0.04 part of binder, 0.1-0.5 part of inorganic fertilizer, 0.5-2 parts of pH regulator, 30-44 parts of seed mixture and 150-200 parts of water, wherein the seed mixture is prepared from the following plant seeds in parts by weight: 10-12 parts of rhizome-type festuca arundinacea, 2-4 parts of Trifolium repens, 3-5 parts of Medicago sativa, 2-3 parts of bermuda grass, 2.5-4 parts of Indigofera pseudotinctoria, 4-6 parts of magnolia multiflora, 3-5 parts of cassia corymbosa, 2-3 parts of amorpha fruticosa and 1-2 parts of cosmos bipinnatus. Through carrying out spray-seeding by using the substrate disclosed by the invention, an imported-soil base course with the strength of 0.25MPa or more can be formed, the rainfall erosion resistance after 45-day plain forming is not lower than 120mm/h, and a shrub-dominated and grass-shrub-combined ecological plant community with the rainfall erosion resistance not lower than 200mm/h is formed in half a year to a year.