We have focused on the DNA strand exchange reaction (SER) as a format of a single-base mismatch detection. We employed a partially double-stranded (PDS) DNA probe having a single-stranded (ss) portion. We could rapidly recognize a single-base mismatch by the SER rates between the PDS probes and target ss DNAs. In the presence of cationic comb-type copolymers (PLL-g-Dex) and betaine, the PDS probes could clearly discriminate between A:T matched and A:G mismatched targets. Furthermore, the PDS probes could discriminate among homo- and hetero-type alleles.