To improve human hepatitis B virus (HBV) polymerase expression in E. coli, we introduced His6 tag to the C-terminus of HBV polymerase. This expression vector, pMPH can direct expression of HBV polymerase with maltose binding protein (MBP) at its N-terminus and a His6 tag at its C-terminus. This allows for the purification of full-size HBV polymerase by a sequential two-step procedure on amylose resin and Ni2+-NTA resin. When MBP/PoI/His fusion protein was purified using amylose resin affinity chromatography, DNA-dependent DNA polymerase (DDDP) specific activity was 3.8 fold higher, and RNA-dependent DNA polymerase (RDDP) specific activity was 2.3 fold higher than those of MBP/PoI fusion protein. Based on these data, the fusion MBP/PoI protein with His6 tag increased the stability of polymerase and prevented its degradation. Since His6 tag is chelated Ni2+-NTA resin, amylose column-purified recombinant protein was reloaded on the second Ni2+-NTA column. The polymerase activity of this Ni2+-NTA column-purified MBP/PoI/His fusion protein was higher than that of amylose column purified MBP/PoI/His fusion protein or MPB/PoI fusion protein. The DDDP specific activity was 16.5 fold higher, and the RDDP specific activity was 5.0 fold higher than that of MBP/PoI fusion protein.