Nitrogen-doped biochar (N-BC) is extensively applied in agriculture, but its potential risks have not been systematically evaluated. In this study, we investigated the phytotoxicity of N-BC and the detoxification strategy of wheat. Results indicated that the formation of reactive oxygen species (ROS) in biochar (BC) was derived from environmentally persistent free radicals (EPFRs). However, N-doping induced the generation of heterocyclic-N and O-containing functional groups on the N-BC surface, which were critical factors for ROS production in N-BC instead of EPFRs. More importantly, heterocyclic-N and O-containing functional groups possessed a stronger ability for ROS generation, causing more severe phytotoxicity (ANOVA, P < 0.05). To counteract the phytotoxicity caused by N-BC, the detoxification strategy of wheat, such as the increase of antioxidant enzyme activities and photosynthesis pigments, was activated to alleviate the stress induced by ROS in vivo (ANOVA, P < 0.05). A split-root experiment demonstrated that N-BC-triggered detoxification of wheat proceeded by promoting the release of amines and/or producing new root exudates, thereby recruiting a group of beneficial microbiota with disease-resisting and growth-promoting (including Cyanobacteria, Bacteroidetes, and Firmicutes) properties to accumulate in wheat rhizospheres. These findings provide a novel perception to assess the safe application of N-BC on agroecosystems.