The invention discloses a tissue culture method of a dioscorea opposita stem with axillary buds. A dioscorea opposita stem with axillary buds is selected as an explant, and through a series of operations of bud induction, mulitiplication and rooting culture, a dioscorea opposita test-tube plantlet is finally formed. The method includes medium components for a plurality of stages of cultivation and a processing method, and a complete tissue culture seedling can be acquired within about twenty days. Through the method, dioscorea opposita tissue culture seedling can be acquired through the induction of the explant of the dioscorea opposita stem with axillary buds, so indoor mass propagation of the test-tube plantlets is feasible, and breeding and industrial production of dioscorea opposita tuber can be realized. The tissue culture method has the characteristics of high propagation coefficient, fast propagation speed, high propagation efficiency and good genetic stability and is free of the influence of environment conditions, and the seedlings have fewer virus.