To explore the enzymatic route of cefatrizine synthesis, a-amino acid ester hydrolase (AEH) gene was cloned from the whole genome of Xanthomonas rubrillineans, and expressed heterologously in Escherichia coli BL21 (DE3). The effects of temperature, pH and substrates’ molar ratio upon the transformation yield of cefatrizine by purified recombinant AEH were investigated. The monomer of AEH was determined as 70 kDa by SDS-PAGE. The optimal pH and temperature reaction were (6.0±0.1) and 36 °C for cefatrizine synthesis. The transformation yield was 64.3% under 36 °C, pH (6.0±0.1), when the concentrations of two substrates were about 30 mmol/L (7-ATTC) and 120 mmol/L (HPGM×HCl), respectively, and the enzyme consumption was 22 U/mL. The results pave the way for optimization of the industrial enzymatic synthesis of cefatrizine.