Phthalates (PAEs), a category of plasticizers released from plastic products, have been widely detected in various environmental media and pose potential ecological risks to humans. Although the exposure risks of PAEs to organisms have been studied, the differences in the interactions between PAEs with different side chain lengths and biomolecules remain poorly understood at molecule levels. In this study, three commonly used PAEs (dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP)) were employed to investigate the influence of their side chain lengths on interactions with catalase (CAT), a key antioxidant enzyme. The effects of PAEs on CAT enzyme activity and their interaction mechanisms were investigated using multi-spectral technique and molecular docking techniques. The results indicate that the order of reduced enzyme activity by PAEs is DMP >
DEP >
DBP, which inversely correlates with the alkyl chain length of PAEs. Molecular docking analysis reveal that DBP failing to bind to the central cavity of CAT likely contributes to its minimal impact on enzyme activity. The multiple spectrums demonstrate that the binding affinity of PAEs to CAT and the changes of CAT conformational structure align with the observed decline in enzyme activity as alkyl chain length increased. Since enzyme activity ties to its structure, the structural alterations in CAT induced by PAEs would inevitably affect its functional expression in vivo. This study offers a comprehensive assessment on the possible toxicity of PAEs with different side chain lengths at the molecular levels, providing insights into their ecological risks.