The invention discloses a transplanting and acclimating method of dendrobium officinale tissue culture seedlings. Sawdust, peanut shells and water are fully mixed in the mass ratio of 1:1.5:2-1:2:2 and then are fermented to serve as a culture substrate; the substrate is paved into a seedbed on which shallow ridges are formed through kicking or holes are dug; the rooted tissue culture seedlings are picked out of culture bottles, and an agar medium is washed clean with clean water; the seedlings are spread flatly to dry water on the surfaces of the plants, and the tissue culture seedlings are fixedly planted in the shallow ridges or holes; a 50% sunshade net is used for shading, the air temperature is controlled to be 10-30 DEG C, the ground temperature is controlled to be 12-28 DEG C, and the seedlings are watered twice in the morning and at night respectively; 3-6 days after transplanting, the substrate is backfilled, the tissue culture seedlings are planted, and the survival rate is 95% or above 15 days after planting. The method reduces the step of hardening-seedling in the bottles in the transplanting and acclimating processes of dendrobium officinale, simplifies procedures, speeds up fibrosis of the tissue culture seedlings, and enhances plant nutrition type change. In addition, the method improves the adaptive capacity of the dendrobium officinale tissue culture seedlings to field environment in a short time and increases the survival rate.