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Background  Raising nitrogen use efficiency of crops by improving root system architecture is highly essential not only to reduce costs of agricultural production but also to mitigate climate change. The physiological mechanisms of how biochar affects nitrogen assimilation by crop seedlings have not been well elucidated.    Results  Here, we report changes in root system architecture, activities of the key enzymes involved in nitrogen assimilation, and cytokinin (CTK) at the seedling stage of cotton with reduced urea usage and biochar application at different soil layers (0–10 cm and 10–20 cm). Active root absorption area, fresh weight, and nitrogen agronomic efficiency increased significantly when urea usage was reduced by 25% and biochar was applied in the surface soil layer. Glutamine oxoglutarate amino transferase (GOGAT) activity was closely related to the application depth of urea/biochar, and it increased when urea/biochar was applied in the 0–10 cm layer. Glutamic-pyruvic transaminase activity (GPT) increased significantly as well. Nitrate reductase (NR) activity was stimulated by CTK in the very fine roots but inhibited in the fine roots. In addition,  AMT1;1, gdh3 , and  gdh2  were significantly up-regulated in the very fine roots when urea usage was reduced by 25% and biochar was applied.    Conclusion  Nitrogen assimilation efficiency was significantly affected when urea usage was reduced by 25% and biochar was applied in the surface soil layer at the seedling stage of cotton. The co-expression of  gdh3  and  gdh2  in the fine roots increased nitrogen agronomic efficiency .  The synergistic expression of the ammonium transporter gene and  gdh3  suggests that biochar may be beneficial to amino acid metabolism.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12870-021-03026-1.

Biochar induced improvement in root system architecture enhances nutrient assimilation by cotton plant seedlings