A pyrene-labeled uridine (UPy) monomer for a pyrrolidinyl peptide nucleic acid with an alternating proline/2-aminocyclopentanecarboxylic acid backbone (acpcPNA) was synthesized and incorporated into the PNA. The UPy base in acpcPNA could specifically recognize the base A in its complementary DNA strand as determined by thermal denaturation (Tm) experiments. The fluorescence of the UPy-containing single-stranded acpcPNA was very weak in aqueous buffer. In the presence of a complementary DNA target, the fluorescence was enhanced significantly (2.7–41.9 folds, depending on sequences). The fluorescence enhancement was specific to the pairing between UPy and dA, making the UPy-modified acpcPNA useful as a hybridization-responsive fluorescence probe for DNA-sequence determination.