AbstractWe have prepared two fluorescent DNA probes—UDBF and UDBT, containing 2‐ethynyldibenzofuran and 2‐ethynyldibenzothiophene moieties, respectively, covalently attached to the base dU—and incorporated them in the central positions of oligodeoxynucleotides (ODNs) so as to develop new types of quencher‐free linear beacon probes and investigate the effect of functionalization of the fluorene scaffold on the photophysical properties of the fluorescent ODNs. The ODNs containing adenine flanking bases (FBs) displayed a selective fluorescence “turn‐off” response to mismatched targets with guanine bases; this suggests that these probes could be used as base‐discriminating fluorescent nucleotides. On the other hand, we observed a “turn‐on” response to matched targets when the UDBF and UDBT units of ODNs containing pyrimidine‐based FBs were positioned opposite the four natural nucleobases. In particular, an ODN incorporating UDBT and cytosine FBs has potential use in single‐nucleotide polymorphism typing.