Phthalate esters (PAEs) show physiological toxicity, and the key to reduce toxicity lies in the complete cleavage of side chain ester bonds. Amidohydrolase RhoⅡ is a hydrolytic enzyme acting on ester bond of monoalkyl phthalate esters, but cannot hydrolyze PAEs. RhoⅡ was modified by computer simulation and site-directed mutation to achieve the purpose of PAEs hydrolysis. Results of the molecular docking between RhoⅡ and monobutyl phthalate (MBP) showed that RhoⅡ stabilized the carboxyl group of MBP with R-groups of Lys200 and Arg185, and both monomers of MBP and RhoⅡ formed hydrogen bond interactions. The site-directed mutation indicated that Asp39, Lys127 and Cys160 formed the catalytic triplet of RhoⅡ, which as the active center positioned in the hydrophobic cavity of the enzymes. Additionally, the mutant enzyme F44N was obtained through site-directed mutation and could hydrolyze PAEs, which significantly improved the enzymatic hydrolysis efficiency toward dibutyl phthalate (DBP) and diisobutyl phthalate (DIBP) compared with the original enzyme. After phenylalanine mutated to asparagine, the steric hindrance effect of the enzyme on the substrate was significantly weakened, and the substrate binding cavity of the enzyme increased. DBP and DIBP could bind to the catalytic pocket of the enzyme to achieve ester bond cleavage. The catalytic mechanism was speculated based on molecular docking and mutation. The mutation might affect the substrate binding cavity, and the mutant enzyme could not effectively bind to the monoalkyl phthalate esters. This work performed sequence and structure analysis of RhoⅡ, and verified the catalyzed triplet. By finding the catalytic activity center of RhoⅡ and mutating the key site, the substrate specificity of RhoⅡ was changed. This study hoped that the enzyme resources related to enzymatic catalytic hydrolysis of PAEs could be expanded and promoted the application of related hydrolases.