The invention relates to the technical field of plant culture, in particular to a strawberry test-tube plantlet acclimatizing and transplanting medium and application thereof. The medium comprises a mixture of humus and herb residues, wherein the mass ratio of the humus to the herb residues is 1:1, the herb residues are products obtained by digesting Chinese herbal medicine as raw materials, the Chinese herbal medicine comprises scutellaria baicalensis, folium ginkgo, privet seeds, cordate houttuynia, polygonum capitatum and rhizoma polygonati, and the folium ginkgo is adopted as the main medicine. Strawberry plantlets obtained by the acclimatizing and transplanting medium have multiple leaves, the color of the leaves is green, root systems are enriched, entangled roots are good, the transplanting survival rate of strawberry test-tube plantlets can be remarkably increased, the strawberry fruit yield is remarkably increased, and the number of sicken plantlets is decreased sharply; in addition, broad-leaf humus and herb residues are adopted as raw materials, the operation is easy, the cost is low, the medium is of a great significance in farmer household life improvement, and the strawberry production level is improved to a new level.