[Objective] To investigate the characters of hydroxymandelate synthase (HmaS) gene in Escherichia coli, the activities of HmaS encoded by hmas isolated from Amycolatopsis orientalis and Streptomyces coelicolor were compared. [Methods] The coding sequences of hmas were respectively amplified from the genomic DNAs of A. orientalis and S. coelicolor and were heterologous expressed in E. coli. The expressing proteins were isolated and purified by anion exchange chromatography and gel filtration chromatography, then the enzymatic activity and catalytic properties of HmaS were evaluated. [Results] The activity of HmaSSC2 from S. coelicolor was almost 3.6 times as of A. orientalis. The optimum reaction temperature of HmaSAO is 28 °C with high storage stability in weak alkaline condition. HmaSSC2 has the optimum reaction temperature of 35 °C and maintains high activity between 28 °C and 45 °C. HmaSSC2 functions well under neutral conditions. [Conclusion] The characters of HmaSSC2 from S. coelicolor is more suitable for metabolic engineering of E. coli to produce mandelic acid.