Rice exposed to organic pollutants such as polybrominated diphenyl ethers (PBDEs) usually experiences reduced biomass and increased soluble sugar content. This study showed that 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47) led to increased glucose, fructose, and sucrose in rice leaves, accompanied by decreased photosynthetic rate and biomass. In order to identify the key enzyme that BDE-47 interacted with, a diazirine-alkynyl photoaffinity probe was designed, and photoaffinity labeling based chemoproteomics was conducted. Among all differentially expressed proteins, fructose-1, 6-bisphosphate aldolase (FBA) involved in carbohydrate metabolism was most likely the target protein of BDE-47. Spectral techniques and molecular docking analysis further revealed that the pollutant-protein interaction was driven by hydrophobic force. BDE-47 inhibited FBA catalytic efficiency by competing with its substrate, fructose-1, 6-diphosphate (F-1, 6-P), leading to soluble sugar accumulation, photosynthetic rate decline and biomass reduction. This study unraveled the influencing mechanism of PBDEs on rice by combining the novel photoaffinity labeling-based chemoproteomics with conventional proteomics. The improved knowledge on direct interaction between organic pollutants and proteins will help alleviate the harmful effects of soil pollution on plants.