Phthalic acid esters (PAEs), including dimethyl phthalate (DMP) and dibutyl phthalate (DBP), were widely known by their potential biological toxicity. In this work, the interaction between PAEs and urease was studied by the resonance light scattering spectrum (RLS), average particle size, molecular docking, molecular dynamics simulation, enzyme activity assay and reaction kinetic experiment. Both DMP and DBP could form stable complexes with urease, and enhance the rigidity and stability of urease. The RLS and average particle size of urease were decreased by DMP and DBP. Moreover, the activation mechanism was non-competitive activation mechanism because K_m of urease kept the same and V_max were increased. It could be concluded that PAEs altered the molecular conformation of urease and increased the urease activity by the non-competitive activation mechanism.