The invention discloses a method of shortening seed breeding cycle of a nicotiana tabacum mutant strain YK01, which is characterized by comprising a seedling raising procedure, a transplant procedure, a vegetative period management procedure, a grafting procedure, a reproductive stage management procedure, and a seed recovery procedure. The method specifically comprises the following steps: the growing environment temperature for seedling raising being 5 to 30 DEG C, the oxygen content being 2-21%, the humidity being 50-100%, and the illumination intensity being 1000 to 100, 000 LX; transplanting the cultured nicotiana tabacum mutant strain YK01 strong seedlings to the area at the elevation of 1400-2500 m; after transplanting, the temperature being 5-35 DEG C, the oxygen content being 5-21%, the humidity being 50-90%, and the illumination intensity being 1000-120, 000 LX; after the vegetative period, grafting the seedlings to the area at the elevation of 400-1500 m; after grafting, the growing environment temperature being 5-35 DEG C, the oxygen content being 5-21%, the humidity being 50-90%, and the illumination intensity being 1000-120,000 LX; spraying one thousandth to one hundredth of leaf fertilizer for 0 to 5 times in the florescence; and recovering the mature seeds. The method is simple and convenient to operate, can effectively shorten the seed breeding cycle of the nicotiana tabacum mutant strain YK01, and improves the quality and the yield of the seeds.