This study demonstrated the enzymatic synthesis of D-tagatose from lactose by engineered Escherichia coli. Recombinant vector pET-32a(+)lacZ-araA that contained the b -galactosidase gene (lacZ) and L-arabinose isomerase gene (araA) of Escherichia coli K-12 was constructed and co-expressed in E. coli BL21 (DE3). The recombinant strain could secreting great amount of b -galactosidase and L-arabinose isomerase simultaneously in soluble fraction. A high conversion rate (³45 mol%) of D-tagatose from lactose was obtained under the optimized conditions: 50 °C, pH 7.0, with Mn²⁺ and at a substrate concentration of 25 g/L. This novel approach could be used to convert D-tagatose directly from lactose and reduce the production cost by approximately 90% at almost the same conversion rate compared with other biological methods.