The recombinant plasmid pET28b -ValG was functionally expressed in E.coli BL21（DE3） and the overexpressed glycosyltransferase was purified sequentially by nickel affinity chromatography, ultra-filtration desalination and freeze-drying. Then the purified enzyme was characterized and it showed good stability at 10-40 °C and pH 5-11. The optimum reaction temperature and pH were 30 °C and 8.0, respectively. The metal ions, such as Ca2+, Mg2+, Mn2+ and Co2+, activated the enzyme activity, while Cu2+ and Zn2+ inhibited the enzyme activity instead. The maximum enzyme activity was obtained at substrate and product concentrations of 8 μmol/L and 2 μmol/L, respectively. When sufficient validoxylamine A was added as substrate, the Lineweaver-Burk plot showed that the KmB and Vm were 35.275 μmol/L and 0.153 μmol/(L·min), respectively. The affinity of validoxylamine A was much better than that of the uridine diphosphate glucose (UDPG) to the glycosyltransferase, which made it necessary to study the supply of UDPG in the process of the whole-cell catalysis.