[Objective] To achieve the high-efficiency synthesis of lactose to tagatose in one-step, we cloned β-galactosidase gene (lacZ) and arabinose isomerase gene (araA) from Escherichia coli K-12 genome and co-expressed in E. coli BL21(DE3).[Methods] The araA and lacZ genes were amplified from the E. coli K-12 genome by PCR. The two genes with SD-AS sequence as a linker were cloned into the expression vector pET28a-1 to get the recombinant plasmid pET28a-araA-lacZ, which was transformed into the competent cells of E. coli BL21(DE3) to obtain E. coli BL21/pET28a-araA-lacZ. The synthesis conditions of tagatose by whole cells of E. coli BL21/pET28a-araA-lacZ were optimized and the process was scaled up.[Results] The araA and lacZ genes were efficiently co-expressed in E. coli BL21 simultaneously. The optimal conditions for the synthesis of tagatose by the whole cells of E. coli BL21/pET28a-araA-lacZ were determined:pH 8.0, 50℃, 5 mmol/L Mn²⁺, 0.5 mol/L borate and 0.1% SDS as permeabilizing agent. Under these optimal conditions, the highest yield of tagatose was 24.03±2.03 g/L with a molar conversion rate of 45.67% using 100 g/L lactose as substrate. The yield of tagatose was increased with the increasment of the substrate lactose concentration. The yield of tagatose was 83.81±1.38 g/L with 500 g/L lactose as substrate.[Conclusion] The genes lacZ and araA coding two target enzymes were co-expressed in E. coli BL21 to realize the efficient synthesis of high valued rare sugar tagatose from the cheap substrate lactose in one step. This research has laid a good research foundation for the preparation of low-energy functional sugars by biological methods.