Pyrene-labeled deoxyuracil and deoxyadenine units are useful unnatural nucleobases. These fluorescent nucleobase analogues allow strong interstrand stacking interactions to compensate for a loss of hydrogen bonding and exhibit a range of different emission intensities when they form duplexes with one another. These findings may provide new insights into the design of new probes and nucleobase analogues for applications in molecular biology. For this purpose, we have prepared an hairpin molecular beacon (MB) that incorporates an excimer unit in its closed state, and have utilized lambda(max) changing to discriminate between match and mismatch. This hairpin configuration is attractive because the synthesis of such an MB is relatively simple and inexpensive because it does not require two distinct processes to prepare the fluorophore and quencher.