Intercalation interactions between planar aromatic molecules and double-stranded DNA (dsDNA) relate to not only the structure of the guest molecules but also the structure of the dsDNA. In this letter, we have comparatively studied the intercalation between pyrene and fully matched or mismatched dsDNA using single-molecule force spectroscopy (SMFS). The significant difference in rupture forces, upon pyrene unbinding from 25-mer dsDNA with or without mismatches, is observed at the single-molecule level, indicating the influence of mismatches on the interaction between pyrene and dsDNA. In the analysis of the dynamic force spectra, two transition barriers are revealed for pyrene unbinding from matched sites in dsDNA and for pyrene unbinding from mismatched sites as well. These results suggest that SMFS is a useful single-molecule method for the detection of mismatches in dsDNA by the intercalation of pyrene.