A pot experiment was conducted at Hetian S&T Research Station of Bijie in Guizhou Province, to explore the effect of biochar application rate on nitrogen (N) and potassium (K) uptake in flue-cured tobacco. A factorial design with two factors (3 types of soils × 5 biochar rates) and a completely randomized layout were used in this experiment. Soil samples of yellow soil, yellow brown soil and calcareous soil were collected from tobacco planting field (0-20 cm). The biochar application rate was 0%, 0.1%, 1%, 2.5% and 5.0% (w/w, equivalent to 0, 1.25, 12.5, 31.3, 62.5 t/hm2 of field use). The results showed that: compared with the control (no biochar application), under the biochar application rate of 5.0%, the number of lateral roots per tobacco plant in yellow soil, calcareous soil and yellow brown soil increased by 17.0, 30.7 and 20.3, respectively, 150.0%, 137.4%, and 67.1% higher than that of the control, respectively. With the increase of biochar rate, the N uptake of tobacco roots, stems, leaves and whole plant increased, but the biochar rates for the highest N uptake by tobacco plants in different types of soils were different. With the biochar rate increase, the K content of roots and leaves of tobacco increased, with the maximum increase of 47.8% and 32.3%, respectively, and the K content of roots increased most obviously. The K uptake in roots, stems and leaves of tobacco increased continuously, with the maximum increase of 311.8%, 87.9% and 144.7%, respectively. In conclusion, the application of biochar can significantly increase the number of lateral roots of tobacco plants, and raise the uptake of N and K in tobacco.