4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides are widely used in modern agriculture. Plant root exudates (REs) play an important role in the adsorption, degradation, migration and transformation of pesticides in soil. In the present study, the structural affinity and interaction mechanism between four HPPD inhibitors (HPPDi) and soybean REs were investigated via multispectral technologies and two-dimensional correlation analysis (2D-COS). UV–vis absorption and fluorescence spectra showed that mesotrione, tembotrione, sulcotrione and topramezone effectively quench the intrinsic fluorescence of soybean REs through static quenching. The binding constant K_a revealed that the binding ability of HPPDi to soybean REs takes the following order: mesotrione > tembotrione > sulcotrione > topramezone. According to the thermodynamic parameters, the main interaction force between tembotrione, sulcotrione, topramezone and soybean REs is electrostatic interaction, while the main interaction force is a hydrogen bond or van der Waals force between mesotrione and soybean REs. The conformational changes of REs were attributed to HPPDi by 3D spectral evaluation. FTIR spectroscopy and 2D-COS analysis suggested that soybean REs mainly formed stable complexes with HPPDi through functional groups such as carbonyl, carboxyl, methoxy and nitrate, and the first binding groups were carbonyl and carboxyl. These results provide helpful information for the adsorption and desorption process of environmental pollutants on the surface of plants and soil.