Nitrogen loss in water from farmland has become a highly concerned environment issue due to the different nitrogen-fertilized input, which is the main cause of agricultural non-point source pollution in the Lake Basin. However, characteristics of nitrogen forms loss from farmland and how the root system responds is lacking. The effectiveness of 100–0% (T1), 75–25% (T2), 50–50% (T3), 25–75% (T4), and 0–100% (T5) nitrate-ammonium nitrogen fertilizer treatment was investigated to determine nitrogen loss in water and root morphological parameters of tobacco in Mile and Chengjiang Counties. Two sites result suggested that: Compared with the T1, T4, and T5 treatments, the TN loss of the T2 and T3 treatments in runoff was reduced by 4.67–27.32%, 11.85–23.20%, and 9.56–31.43%, respectively. Similar results in the nitrogen loss of infiltration were observed with that from runoff. Correlation analysis suggested that the root biomass was negatively correlated with the nitrogen loss. T2 and T3 treatments were larger than those of the T1, T4, and T5 treatments in terms of root biomass, root surface area, and root spatial distribution. This study suggested that 50–50% nitrate-ammonium nitrogen fertilizer can facilitate the root growth of tobacco and reduce nitrogen loss, which provides a reference for agricultural sustainable development.