Selective recognition of double strands DNA (dsDNA) has been a research hot spot in molecular biology and biomedicine for a couple decades. Based on the selective interaction between natural nucleic acid/synthetic molecular ligands and dsDNA, gene diagnosis, gene therapy and gene editing would be realized. Hairpin oligopolyamide is a molecular ligand with excellent cellular permeability and nucleases-resistance which can target dsDNA sequence with high affinity and specificity at minor groove. This paper reviews the binding properties and biomedical applications of hairpin oligopolyamide targeting dsDNA, which provide references for further design and application of hairpin oligopolyamide.