The invention discloses an ex vitro rooting method of sugarcane test tube plantlets, which comprises the following production procedures of: (1) establishing a sterile culture system; (2) proliferating and expanding; (3) transplanting single rootless test tube plantlets to a nursery for ex vitro rooting and seedlings growing; and (4) transplanting to a large field. With the method, the sterile culture rooting procedure in a culture room is omitted, the rooting culture operations and the rooting culture conditions of the sugarcane test tube plantlets are simplified, and the complex operating procedures of culture medium preparation, high pressure sterilization, sterile tube transfer on a clean bench, constant temperature and humidity culture in the culture room and the like are omitted. The ex vitro rooting method of the rootless sugarcane test tube plantlets is simple in operation and can be operated in a natural environment, the production cost of sugarcane test tubes is reduced, andthe production efficiency is improved.