This study was conducted to explore effects of biochar-based fertilizer on physiology and nitrogen uptake of flue-cured tobacco and nitrogen balance in the yellow soil－flue-cured tobacco system, which will provide certain scientific guidance for optimizing nitrogen management in the yellow soil of the tobacco-planting areas in Guizhou and reducing pollution in the ecological environment of the farmland of tobacco-planting areas in Guizhou. A field experiment, carried out with flue-cured (Yunyan 87)in yellow soil for the study, was designed to have three treatments, i.e. no nitrogen (CN), conventional fertilizer (CF), biochar-based fertilizer (BF). Results show that Treatment BF significantly increased soil urease activity, root length and surface area, glutamine synthase and glutamate synthetase activities in leaves, and nitrogen uptake of flue-cured tobacco. Treatment BF was significantly 35.06%and 14.24% higher than Treatments CN and CF in nitrogen uptake of flue-cured tobacco, respectively. Treatment BF was the highest in activity, volume and dry biomass of root and yield of flue-cured tobacco, and significantly higher than Treatment CN. Nitrogen surplus was obviously detected in the yellow soil－flue-cured tobacco system in all the treatments, the nitrogen input was mainly the initial soil inorganic nitrogen, while the nitrogen output mainly consisted of soil inorganic nitrogen residue and nitrogen uptake of flue-cured tobacco. Treatment BF increased inorganic nitrogen residue in the 0-20 cm soil layer. Moreover, Treatment BF significantly decreased nitrogen apparent loss by 44.42 kg·hm^–2, and decreased nitrogen surplus by 37.70 kg·hm^–2, and significantly increased nitrogen apparent recovery efficiency by 16.15%. Therefore, the reasonable application of biochar-based fertilizer is an effective way to optimize management of nitrogen in the yellow soil and protect ecological environment of tobacco-planting areas in Guizhou.