Display omitted •The crystal structure of hnRNP A2/B1 RRMs dimer in complex with U-shaped conformation of ssDNA has been determined.•hnRNP A2/B1 RRMs homodimerise in solution by binding to pre-generated U-shaped DNA, either in ssDNA or dsDNA.•hnRNP A2/B1 RRMs homodimer induced by U-shaped ssDNA binding in solution exists a “Dimer A2” protein dimer interface.•The dimer interface formed in solution biochemically which depicts a potential functional state of hnRNP A2/B1. Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been identified as a nuclear DNA sensor. Upon viral infection, hnRNP A2/B1 recognizes pathogen-derived DNA as a homodimer, which is a prerequisite for its translocation to the cytoplasm to activate the interferon response. However, the DNA binding mechanism inducing hnRNP A2/B1 homodimerization is unknown. Here, we show the crystal structure of the RNA recognition motif (RRM) of hnRNP A2/B1 in complex with a U-shaped ssDNA, which mediates the formation of a newly observed protein dimer. Our biochemical assays and mutagenesis studies confirm that the hnRNP A2/B1 homodimer forms in solution by binding to pre-generated ssDNA or dsDNA with a U-shaped bulge. These results depict a potential functional state of hnRNP A2/B1 in antiviral immunity and other cellular processes.