Although distance dependence of Förster resonance energy transfer (FRET) is well-studied and FRET has been extensively applied as "molecular ruler", only limited examples of orientation-dependent FRET have been reported. To create a robust FRET system that precisely reflects the orientation between donor and acceptor, donor and acceptor fluorophores were introduced into a DNA via a D-threoninol scaffold. Strong stacking interactions among intercalated dyes and natural base-pairs suppress free movement of the dyes, clamping them in the duplex in a fixed orientation. Pyrene and perylene were used as donor and acceptor, respectively, and both the distance and orientation between these dyes were systematically controlled by varying the number of intervening AT pairs from 1 to 21 (corresponding to two turns of helix). FRET efficiency determined from static fluorescence measurement did not decrease linearly with the number (n) of inserted AT pairs but dropped significantly every 5 base pairs (i.e., n = 8, 13, and 18), corresponding to a half-turn of the B-type helix. This clearly demonstrates that FRET efficiency reflects the orientation between pyrene and perylene. We also measured time-resolved fluorescence spectroscopy with a streak camera and successfully observed the time-course of the energy transfer directly. As expected, the FRET efficiencies determined from the lifetime of pyrene emission were in good agreement with static measurements. Theoretical calculation of FRET efficiency assuming that the DNA duplex is a rigid cylinder with B-type geometry coincided with the experimental results. We believe that our method of using d-threoninol will contribute to further development of FRET-based measurement techniques.