Only a few esterases have been used for the synthesis of optically pure fungicide. For example, (R)-metalaxyl synthesized using esterase-involved bioreaction displays fungicide activity, whereas (S)-enantiomer is redundant. However, the biosynthesis of (R)-metalaxyl is currently hampered by the lower activity, selectivity and thermostability of esterase. Therefore, to obtain a better biocatalyst, several esterase genes were cloned from Pseudochrobactrum asaccharolyticum WZZ003. The esterase PAE07, among eight enzymes, was selected because it exhibited the highest hydrolysis activity toward (R,S)-DMPM. The DNA and amino acid sequence analysis suggested that PAE07 is a new member of lipolytic enzyme family V. The enzymatic properties of PAE07 toward (R,S)-DMPM and model substrate (p-nitrophenyl acetate) were investigated. PAE07 was found to be a highly active esterase with excellent enantioselectivity. The reaction conditions including temperatureand pH were optimized, and the effects of metal ions, organic solvents and detergents were also investigated. Results indicated that PAE07 is a competitive candidate for (R)-metalaxyl manufacturing.