The invention relates to a micro-ecological root system regulator special for flue-cured tobacco in an upland field and an application method of the micro-ecological root system regulator and belongs to the technical field of agricultural fertilizers. The micro-ecological root system regulator special for flue-cured tobacco in the upland field comprises the following components in parts by weight: 30-35 parts of sodium alga acid, 20-25 parts of compound sodium nitrophenolate, 20-25 parts of magnesium sulfate, 10-15 parts of boric acid, 10-15 parts of rooting powder, 10-15 parts of expansin and 1-2 parts of manganese sulfate. The regulator is dissolved with water and applied by using the methods of spraying leaves at a nursery stage, performing root-irrigation at a transplanting period or film breaking, releasing seedlings and irrigating holes. According to the regulator provided by the invention, the various components are matched with one another and act together, so that the micro-ecological environment of the root system of the flue-cured tobacco in the upland field can be effectively improved, and the effects of rapidly promoting root cell division and primordium germination of lateral roots, promoting root cell wall loosening, increasing the quantity of the lateral roots and accelerating growth of young roots are achieved. Meanwhile, soil granulation structure formation is promoted, multiple microbes in soil are activated, nutrient releasing is accelerated, the weakly acid environment of the soil is regulated, trace elements are supplemented, the root soil micro-ecology is comprehensively regulated, and early growth and quick tiller of the root system of the flue-cured tobacco can be promoted.