The invention discloses a culture medium for culturing dendrobium officinale tissue cultured seedling and a method for preparing the culture medium, and belongs to the technical field of dendrobium officinale planting. The culture medium for culturing the dendrobium officinale tissue cultured seedling comprises the following components: mashed potatoes, applesauce, banana sauce, white sugar, ammonium nitrate, potassium nitrate, potassium dihydrogen phosphate, boric acid, amino acids, vitamins, IBA, GA, 6-BA and pure water. According to the invention, growth hormones IBA, GA and 6-BA are added to the culture medium for culturing dendrobium officinale tissue cultured seedling, so that the roots, buds and leaves can be promoted to grow quickly; and in addition, the culture medium is rational in component match and comprehensive in nutrition, and can shorten the culturing period of the dendrobium officinale and increase the survival rate of the transplanted dendrobium officinale to 98.75%. The method for preparing the culture medium for culturing the dendrobium officinale tissue cultured seedling, provided by the invention can shorten the disinfection time, improve the sterilization effect reliability and the industrialized production efficiency, and reduce the production cost. The method for preparing the culture medium for culturing the dendrobium officinale tissue cultured seedling is simple to operate and liable to realize the industrialized, normalized and standardized production.