Bst DNA polymerase is a key enzyme in isothermal amplification reaction. However, due to the patent restrictions, most of the Bst DNA polymerase used in domestic laboratories need to be imported abroad, which is expensive and needs long distance transportation. Therefore, it is urgent to produce Bst DNA polymerase for isothermal amplification detection technology independently. From constructing a plasmid containing Bst Fragment, Bst DNA polymerase was prepared on a large scale by prokaryotic expression system followed by His-tag affinity purification method. The purified Bst DNA polymerase possessed the advantages such as low cost, high activity, and strong specificity in the isothermal amplification reaction. The activity of Bst DNA polymerase was optimized for specific DNA templates by detecting the products of amplification. At the same time, the optimal amplification reaction conditions of the enzyme were finally selected，which were 60 mM K+，30 mM（NH4）2SO4，and pH 9.0. With reference to the relevant patents, the obtained Bst DNA polymerase was able to detect mycoplasma pneumonia and Chlamydia pneumonia quickly and accurately through isothermal amplification reactions. In conclusion, a low cost enzyme was provided for isothermal amplification detection of related pathogen DNA, which can be potentially used in the primary medical care.