Optical imaging in the near-infrared (NIR) range enables detecting ligand-receptor interactions and enzymatic activity in vivo due to lower scattering and absorption of NIR photons in the tissue. We designed and tested prototype NIR fluorescent oligodeoxyribonucleotide (ODN) reporters that can sense transcription factor NF-κB p50 protein binding. The reporter duplexes included donor NIR Cy5.5 indodicarbocyanine fluorochrome linked to the 3′ end of the first ODN and NIR acceptor fluorochromes (indodicarbocyanine Cy7 or, alternatively, a heptamethine cyanine IRDye 800CW) that were linked at the positions +8 and +12 to the complementary ODN that encoded p50 binding sites. Both Cy7 and 800CW fluorochromes were linked by using hydrophilic internucleoside phosphate linkers that enable interaction between the donor and the acceptor with no base-pairing interference. We observed efficient fluorescence resonance energy transfer (FRET) both in the case of Cy5.5–Cy7 and Cy5.5–800CW pairs of fluorochromes, which was sensitive to the relative position of the dyes. Higher FRET efficiency observed in the case of Cy5.5–Cy7 pair was due to a larger overlap between the ODN-linked Cy5.5 emission and Cy7 excitation spectra. Fluorescent mobility shift assay showed that the addition of human recombinant p50 to ODN duplexes resulted in p50 binding and measurable increase of Cy5.5 emission. In addition, p50 binding provided a concomitant protection of FRET effect from exonuclease-mediated hydrolysis. We conclude that NIR FRET effect can be potentially used for detecting protein–DNA interactions and that the feasibility of detection depends on FRET efficacy and relative fluorochrome positions within ODN binding sites.