The dual‐labeled oligonucleotide derivative, FAT‐0, carrying 6‐ carboxyfluorescein (FAM) and 6‐carboxytetramethylrhodamine (TAMRA) labels at the 5′ and 3′ termini of the thrombin‐binding aptamer (TBA) sequence 5′‐GGT TGG TGT GGT TGG‐3′, and its derivatives, FAT‐n (n=3, 5, and 7) with a spacer at the 5′‐end of a TBA sequence of T_mA (m=2, 4, and 6) have been designed and synthesized. These fluorescent probes were developed for monitoring K⁺ concentrations in living organisms. Circular dichroism, UV‐visible absorption, and fluorescence studies revealed that all FAT‐n probes could form intramolecular tetraplex structures after binding K⁺. Fluorescence resonance energy transfer and quenching results are discussed taking into account dye–dye contact interactions. The relationship between the fluorescence behavior of the probes and the spacer length in FAT‐n was studied in detail and is discussed.