The invention provides a dendrobium officinale cultivation grain substrate and a preparation method thereof. The dendrobium officinale cultivation grain substrate is prepared from raw materials of, based on weight rate, 45%-55% of abandoned edible mushroom residue, 3%-6% of Chinese herbal medicine waste residue, 2%-5% of chestnut shell, 4%-8% of peanut shell, 10%-15% of mulberry stem, 4%-10% of corncob, 5%-8% of wood chip, 1.0%-2.0% of quick lime, 0.2%-1.0% of urea, 0.2%-1.0% of monopotassium phosphate, 0.1%-0.5% of zinc sulfate, 3.0%-7.0% of clay, 5%-10.0% of sod soil and 0.5%-1.0% of carbendazol wetting powder. The dendrobium officinale cultivation grain substrate can improve disease resistance of dendrobium officinale, and effectively reduces occurrence rate of diseases of root rot, soft rot and the like for the dendrobium officinale. According to the dendrobium officinale cultivated by using the dendrobium officinale cultivation grain substrate, survival rate can reach above 98%, the average plant height can reach up to 78cm one and a half years after the dendrobium officinale is cultivated, the average steam thickness reaches 7.5-8.2mm, polysaccharide content reaches 42.5%, and an average dendrobium officinale yield per mu can reach 580kg.