Accurate DNA synthesis is vital to maintain genome stability and ensure propagation of species. Synthetic errors have far reaching consequences. Therefore, DNA synthesis is remarkably accurate. The high fidelity is mainly achieved through three steps: ① nucleotide selection, which is based on hydrogen, base pair shape, or some other elements; ② 3'→5' exonuclease proofreading, which removes mis-incorporated nucleotides in cis or trans; ③ repair process, which could correct mismatched nucleotides escaping from proofreading, such as mismatch repair, excission repair, homologous recombination repair, and translesion DNA synthesis. Because all polymerases are suitable targets for anticancer or antiviral drugs, their fidelity is involved in drug resistance and side effects. Understanding the molecular basis of synthesis fidelity is of vital importance. In this review, the fidelity mechanisms of DNA synthesis will be discussed in detail. Furthermore, their application perspective was discussed.