DNA sequencing using thermostable DNA polymerase is very valuable in molecular biology. From a specific strain of Bacillus stearothermophilus, the gene of the Bst DNA polymerase large fragment which had no 5'-3' exonuclease activity was cloned and recombined into an expression vector pYZ23,and the constructed plasmid was introduced into Escherichia coli JF1125,then the expressed protein was isolated and purified. The genetic engineered product shows characteristics similar with the purified natural Bst DNA polymerase large fragment. It will benefit the wide application of Bst DNA polymerase and the study of the relationship between the structure and function of Bst DNA polymerase.