Three fluorene-labeled 2′-deoxyuridines that differ in terms of their linkers—U^F (without linker), U^FL (with ethynyl linker), and U^DF (with diethynyl linker)—have been introduced at the central positions of oligodeoxynucleotides to examine the effects that their linkers have on the fluorescence emission properties upon duplex formation with fully matched and single-base-mismatched targets. Here, we describe the influence of the linkers on the emission behavior, the intramolecular electron transfer between the fluorene moiety and the uracil base after photoexcitation, and the structural stability upon duplex formation. The probe containing the U^FL residue (with an ethynyl linker) and cytosine residues as flanking bases exhibited the greatest fluorescence turn-on selective behavior toward the perfectly matched target.