The invention discloses a breeding method of a single-grain beet high-resistance pairing sterile line. The method comprises the steps of selecting high-resistance and single-grain beet base material, and carrying out summer seeding to cultivate a mother root; carrying out spatial isolation planting, inspecting the grain fertility of all single plants, separating single-grain sterile plants from single-grain fertile plants, and respectively listing to obtain seeds of the sterile plants and corresponding seeds of the fertile plants; carrying out centralized isolated hybridization on plant pairs, and harvesting to obtain first-filial generation F1 strain seeds; carrying out backcross selection by a backcross transformation method, and selecting the strain material with the single fertility rate higher than 95% in the sterile line; indentifying and selecting the cultivated single-grain paired male sterile material on a beet spring sowing disease garden land or a beet seed collecting disease garden land; carrying out disease resistance screening, selecting and remaining the plants with resistance, and harvesting the seeds in pairs. Compared with the prior art, the breeding method has the beneficial effects that the single-grain sterile line with high purity can be cultivated, the single grain rate reaches 99%, and the sterility rate reaches 95%. After being indentified, selected and cultivated on the disease garden land for a plurality of years, the single-grain beet pairing sterile line is high in resistance.