Extensively applied glufosinate (GLU) will trigger molecular alterations in nontarget tea plants (Camellia sinensis), which inadvertently disturbs metabolites and finally affects tea quality. The mechanistic response of tea plants to GLU remains unexplored. This study investigated GLU residue behavior, the impact on photosynthetic capacity, specialized metabolites, secondary pathways, and transcript levels in tea seedlings. Here, GLU mainly metabolized to MPP and accumulated more in mature leaves than in tender ones. GLU catastrophically affected photosynthesis, leading to leaf chlorosis, and decreased Fv/Fm and chlorophyll content. Physiological and biochemical, metabolomics, and transcriptomics analyses were integrated. Showing that GLU disrupted the photosynthetic electron transport chain, triggered ROS and antioxidant system, and inhibited photosynthetic carbon fixation. GLU targeted glutamine synthetase (GS) leading to the accumulation of ammonium and the inhibition of key umami L-theanine, causing a disorder in nitrogen metabolism, especially for amino acids synthesis. Interestingly, biosynthesis of primary flavonoids was sacrificed for defensive phenolic acids and lignin formulation, leading to possible losses in nutrition and tenderness in leaves. This study revealed the defense intricacies and potential quality deterioration of tea plants responding to GLU stress. Valuable insights into detoxification mechanisms for non-target crops post-GLU exposure were offered. Environmental implicationGlufosinate, the world's second-largest herbicide with increasing production amidst rising challenges of glufosinate-resistant weeds, is extensively utilized in tea plantations. Glufosinate threatens nontargeted tea safety and consumer health by disturbing crop metabolism. This investigation delves into the intricate interplay between glufosinate and the morphological, physiological, metabolomic, and transcriptomic responses of tea plants. The disruption of nitrogen metabolism and secondary metabolite synthesis of nontargeted crops from the contaminant underscores the potential ecological consequences. Moreover, this study provides new information for the screening of biomarkers of glufosinate stress-responsive metabolites or genes and the detoxification mechanism of resistance to glufosinate stress.