The invention provides a tissue culture method for one-step seed formation by using Jingzhou pinellia ternate tissue-culture cluster seedlings. The method includes the following steps that 1, with-leaf petioles are taken as explants; 2, the explants are inoculated into a seed formation medium for culture to form bulbils and/or apical buds, and then the buds grow into seedlings after maturation, wherein the seed formation medium includes 1.3-1.7 mg/L of 1/2MS + IBA, 0.2-0.8 mg/L of NAA, 0.05-0.15 mg/L of TDZ, 3% of sucrose and 0.7% of agar. The with-leaf petioles of the Jingzhou pinellia ternate tissue-culture cluster seedlings serve as the explants, the special culture medium is adopted, formation of the bulbil and apical bud structures is facilitated, the bulbil and apical bud structures are similar to seed structures, the bulbils and apical buds can grow into the normal seedlings after maturation, the germination rate and the callus rate are high, no seedling exercising is required, seeds can be directly sowed, the survival rate reaches 100%, the quality of the seedlings is guaranteed, and scale production is achieved.