To provide theoretical reference for the efficient use in cultivation of Isatis indigotica, field experiment was carried to analyze the indigo, indirubin, total alkaloids, (R,S)epigoitrin and polysaccharide of I. indigotica from five different areas by using seven nitrogen treatments with three repeats, which were nitrogen control (CK), NO₃^-N, NH₄⁺N, NH₄⁺N/NO₃^-N=75/25, NH₄⁺N/NO₃^-N=50/50, NH₄⁺N/NO₃^-N=25/75 and CO(NH₂)₂, respectively. The results showed that nitrogen treatment was beneficial to improve its indigo of I. indigotica leaves from Yuncheng Shanxi and Shangluo Shaanxi. Meanwhile, the nitrogen treatment could also improve the total alkaloids of Bozhou Anhui and Shangluo Shaanxi. The content of indigo, indirubin and total alkaloids were all increased in the group of Yuncheng Shanxi with NH₄⁺N/NO₃^-N=50/50, as well as Yuncheng Shanxi and Shangluo Shaanxi with amide nitrogen. Compared with control, nitrogen treatment could effectively improve the (R,S)epigoitrin content of I. indigotica roots from Zhangye Gansu and Shangluo Shaanxi, and the polysaccharide content of I. indigotica roots from Bozhou Anhui. The (R,S)epigoitrin content of I. indigotica roots from Fuyang Anhui was much higher than that of others in any treatment. These results indicated that there are many differences among the responses of nitrogen treatment in different areas. Therefore, we could improve the contents of active components of the I. indigotica by applying costeffective nitrogen treatment according to the resource of I. indigotica and fertilizerrequired rhythm.