Diclofop-methyl, an aryloxyphenoxypropionate (AOPP) herbicide, is a chiral compound with two enantiomers. Microbial detoxification and degradation of various enantiomers is garnering immense research attention. However, enantioselective catabolism of diclofop-methyl has been rarely explored, especially at the molecular level. This study cloned two novel hydrolase genes (dcmA and dcmH) in Sphingopyxis sp. DBS4, and characterized them for diclofop-methyl degradation. DcmA, a member of the amidase superfamily, exhibits 26.1-45.9% identity with functional amidases. Conversely, DcmH corresponded to the DUF3089 domain-containing protein family (a family with unknown function), sharing no significant similarity with other biochemically characterized proteins. DcmA exhibited a broad spectrum of substrates, with preferential hydrolyzation of (R)-(+)-diclofop-methyl, (R)-(+)-quizalofop-ethyl, and (R)-(+)-haloxyfop-methyl. DcmH also preferred (R)-(+)-quizalofop-ethyl and (R)-(+)-haloxyfop-methyl degradation while displaying no apparent enantioselective activity towards diclofop-methyl. Using site-directed mutagenesis and molecular docking, it was determined that Ser175 was the fundamental residue influencing DcmA's activity against the two enantiomers of diclofop-methyl. For the degradation of AOPP herbicides, DcmA is an enantioselective amidase that has never been reported in research. This study provided novel hydrolyzing enzyme resources for the remediation of diclofop-methyl in the environment and deepened the understanding of enantioselective degradation of chiral AOPP herbicides mediated by microbes.