The present invention provides a high-fidelity Pfu DNA polymerase mutant, which is characterized in that, on the basis of wild-type Pfu DNA polymerase, mutations of 1, 3 or 6 amino acid sites are carried out, wherein the mutation sites are as follows: Loci selected: Y403A, P411H, V438K, A741S, R757Q, S768K. And disclosed its coding DNA, and its application in NGS. The high-fidelity Pfu DNA polymerase mutant of the present invention has ultra-high fidelity and high amplification efficiency, can be stably and efficiently applied to the library enrichment process in next-generation sequencing, effectively improves the quality of library sequencing, and reduces the base mismatch rate. It can be used for precise analysis and interpretation of genetic information.