The invention discloses a method for screening root media by using tobacco sterile seedlings. The method comprises the following steps: A, sterilizing tobacco seeds; B, cultivating the sterile seedlings; C, shearing the sterile seedlings; D, allocating a plurality of root media to be screened; and E, screening the optimal root media. The method is simple in process, labor-saving and time-saving, economic and reliable, and can shorten the screening period effectively, simplify the screening program, and save the screening material; and the sterile seedlings materials are obtained by seeds in a culture vessel directly, and enter the stage of root screening directly, which omits the trouble of cultivating adventitious buds as compared with the conventional screening test, so that manpower, material resources and time are saved. The tobacco sterile seedlings are adopted to screen the media, and the tobacco sterile seedlings screening can synchronize with the callus induction, so that the test schedule is speeded up. Besides, the induction effect of the method is consistent with that of a method for screening root media by using adventitious buds.