The invention provides a preparation method of a seedling growing substrate for greenhouse planting, wherein the preparation method comprises the following step: preparing materials: saw dust, cow dung, chicken manure, straw powder, crushed corn cob, perlite, vermiculite, plant ash, belvedere fruit, pine needle, thunder god vine, radix stemonae, sodium alginate, potassium fulvate, sodium dodecyl sulfate, sodium carboxymethyl starch, a sodium chloride water solution with a mass percent of 15 percent, and fine earth; and the preparation method specifically comprises the following steps: A, fermenting raw materials, namely, after uniformly stirring the saw dust, the cow dung, the chicken manure, the straw powder, the crushed corn cob and the fine earth, controlling the water content to be 57-62 percent, adding a rotting-promoting agent; B, preparing a traditional Chinese medicine extraction solution; C, taking the plant ash to obtain a plant ash leaching liquor; D, adding the sodium alginate, the potassium fulvate, the sodium dodecyl sulfate and the sodium carboxymethyl starch in the sodium chloride water solution with a mass percent of 15 percent; E, soaking the vermiculite in the traditional Chinese medicine extraction solution; and F, proportioning the fermented materials obtained in the step A, the soaked pearlite obtained in the step D, and the vermiculite obtained in the step E according to a ratio of 8:1:1. The seedling growing substrate is simple in proportioning, convenient to use, good in seedling growing effect, developed in seedling root system, good in water-retaining property and rich in nutritional ingredients.