The phenylurea herbicide linuron is globally used and has caused considerable concern due to its environmental pollution. In this study, a highly efficient linuron-transforming strain Sphingobium sp. SMB was isolated, and a gene (lahB) responsible for the hydrolysis of linuron to 3,4-dichloroaniline and N,O-dimethylhydroxylamine was cloned from the genome of strain SMB. The lahB gene encodes an amidohydrolase, which shares 20%-53% identity with other biochemically characterized amidohydrolases, except the newly reported linuron hydrolase Phh (75%). The optimal conditions for the hydrolysis of linuron by LahB were determined to be pH 7.0 and 30 °C, and the Km value of LahB for linuron was 37.3 ± 1.2 µM. Although LahB and Phh shared relatively high identity, LahB exhibited a narrow substrate spectrum (specific for linuron) compared to Phh (active for linuron, diuron, chlortoluron, etc.). Sequence analysis and site-directed mutagenesis revealed that Ala261 of Phh was the key amino acid residue affecting the substrate specificity. Our study provides a new amidohydrolase for the specific hydrolysis of linuron.