Abstract: To completely remove the residual host DNA from Escherichia coli in Taq DNA polymerase, this study utilized the pET-15b plasmid for high-efficiency expression of Taq DNA polymerase in Escherichia coli BL21（DE3）, with IPTG induction ensuring high-level expression of the target protein. A purification strategy combining Ni-NTA affinity chromatography and DEAE anion exchange chromatography was employed, successfully yielding the target enzyme with a purity exceeding 95%. PCR detection of the Escherichia coli 16S rRNA gene revealed that the residual host DNA levels in the Taq DNA polymerase purified in this study were comparable to those of commercially available Taq DNA polymerase. Compared to traditional methods such as heat denaturation combined with ammonium sulfate precipitation or single-step Ni-NTA affinity chromatography, the optimized purification process effectively eliminated host DNA residues, providing an efficient and feasible strategy for the preparation of high-purity Taq DNA polymerase.