The disclosure provides a high-fidelity polymerase with preference for gapped DNA and use thereof. The Klenow fragment (KlenDr) derived from Deinococcus radiodurans DNA polymerase I, which has the high-fidelity polymerization characteristics, is independent of 3′-5′ proofreading exonuclease activity, has the preference for binding gapped DNA, and is different from the existing commercial high-fidelity polymerase. Due to the specific affinity of KlenDr to gapped DNA substrate, the 3′ end of the forward primer will not be cut off, and the downstream nucleotide chain is rarely replaced.