i‐Motif (iM) DNA, a cytosine‐rich sequence having a four‐stranded structure, is believed to play an important role in regulating gene expression. In this study, we replaced each of the four thymidine units of the human telomeric iM sequence individually with a 2‐dimethylaminofluorene–substituted 2'‐deoxyuridine derivative (UDAF), then evaluated the formation of the iM structures with respect to pH through melting temperature measurements and circular dichroism, fluorescence, and UV‐Vis spectra. Introduction of the UDAF residue did not adversely affect the formation or stability of the iM structure; one of these strands functioned as an efficient fluorescent probe for iM formation.