The invention discloses a selenium-rich watermelon seedling substrate. The substrate is prepared from, by weight, 40-50 parts of vermicompost, 10-15 parts of vermiculite, 8-10 parts of humus soil, 10-15 parts of molasses fermentation liquor, 3-5 parts of sesames, 3-5 parts of tangerine pith, 3-5 parts of fermentative strains, 20-30 parts of soft sediments with water content of 90%, 15-20 parts of atriplex patens, 3-5 parts of duckweed, 1-2 parts of fosamine ammonium and 10-20 parts of inorganic fertilizer, wherein the fermentative strains comprise microzyme, lactic acid bacteria, actinomycete and bacillus subtilis with the mass ratio of microzyme to lactic acid bacteria to actinomycete to bacillus subtilis being (3-6):(4-6): (2-3):1. The invention further discloses a preparation method of the selenium-rich watermelon seedling substrate. The selenium-rich watermelon seedling substrate is comprehensive in nutritional ingredient and balanced and harmonious in proportion; sufficient and coordinated nutrition can be supplied to watermelons in the whole growth period, and the growth of the watermelons is effectively promoted; the good quality of the watermelons is ensured, the selenium-rich watermelon seedling substrate can also be efficient, and the yield is efficiently improved.