We designed and synthesized sensors for imaging transcription factor–DNA interactions using a complementary pair of 21-base pair long oligonucleotides (ODNs) carrying two internucleoside phosphate-linked cyanine fluorophores that can either engage in Förster's resonance energy transfer (FRET) with fluorescence emission or assemble into a ground state quenched dimer with short fluorescence lifetimes (FL). Cyanine fluorophores were linked to ODNs within the NF-κB binding site. These sensors were tested in the presence of recombinant p50 and p65 NF-κB proteins or constitutively NF-κB activating HeLa cell lysates. By using a coherent light excitation source we followed changes in fluorescence lifetime of the donor (Cy5.5) at the donor's excitation and emission light wavelengths, as well as the acceptor (800CW or Cy7 cyanine fluorophores) in FRET mode. We observed increases in the donor lifetime in both emitting (0.08–0.15 ns) and non-emitting quenched (0.21 ns) sensors in response to protein binding. The measurements of lifetimes in FRET mode in quenched pair-carrying ODN duplex sensors showed significant differences in FL of the acceptor cyanine fluorophore between NF-κB-containing and NF-κB-free samples but not in control sensors with ODN sequences that have decreased binding affinity to NF-κB. We anticipate that the observed effects will be instrumental for developing sensors enabling non-invasive imaging in cells that undergo activation of NF-κB.