Forster resonance energy transfer (FRET) has been used as a molecular ruler to monitor the distance between or within biomolecules. A distance- and orientation-dependent FRET system can be a more powerful tool to investigate biomolecules because of its higher spatial sensitivity. For this purpose, distance- and orientation-dependent FRET pairs have been developed using a DNA scaffold; however, none of them use an isomorphic thymidine analog. Herein, we report the construction of a FRET pair using a fluorescent thymidine analog, T-diox, as the donor and tC as the acceptor. The experimental FRET efficiency was calculated based on steady-state fluorescence spectra and time-resolved fluorescence (lifetime). The calculated values showed a periodical change, which suggests the sensitivity of FRET efficiency to the orientation of fluorophores. For further evaluation of our FRET pair, we compared the theoretical FRET efficiency with the experimental value and observed a relatively high consistency. This is the first color variable FRET system that used a fluorescent thymidine analog, which is expected to expand the applicability of nucleobase-analog FRET pairs.