The invention relates to the technical field of agriculture plantation, and concretely relates to a herbicide used for stevia rebaudiana plantation and a preparation method thereof. The herbicide comprises the following components: Setaria viridis, aconitum sinomontanum nakai, euphorbia, artemisia argyi, bidens pilosa, euphorbia helioscopia, golden larch bark, angled bittersweet, tripterygium wilfordii, radix sophorae flavescentis and rheum officinale; the preparation method comprises the following steps: respectively weighing the components, crushing the components, uniformly mixing the components; adding the components in a soxhlet extractor, adding ethanol, performing ultrasonic-assisted extraction, taking the mixing components to obtain an extract; placing the mixing components in a flask, adding water, performing ultrasonic-assisted heat backflow, filtering the material while hot, repetitively operating for 2 times; merging the obtained extract, a filtrate and a washing lotion, and concentrating the materials to obtain the target product herbicide. The herbicide used for stevia rebaudiana plantation can effectively remove the weeds, and can inhibit germination of weed seeds, and the preparation method is simple, operation is convenient, and the method is suitable for popularization and application.