The incorporation of gold nanoparticles (Au NPs) as quencher modules in fluorescent probes for DNA damage caused by intracellular hydroxyl radicals (HO*) is reported. Au NPs of 15 nm diameter were decorated with DNA oligomers terminating in thiol functions in their 3' positions and possessing 5' fluorophore modifications. The Au NPs, which have high extinction coefficients, functioned as excellent fluorescent quenchers in the fluorophore-Au NP composites. FRET is switched off as a factor of HO*-induced strand breakage in the single-stranded DNAs, restoring the fluorescence of the quenched fluorophores, which can be followed by spectrofluorimetry. In vitro assays with HO*-generating Fenton reagent demonstrated increases in fluorescence intensity with a linear range from 8.0 nM to 1.0 microM and a detection limit as low as 2.4 nM. Confocal microscopic imaging of macrophages and HepG2 revealed that the probe is cell-permeable and intracellular HO*-responsive. The unique combination of good selectivity and high sensitivity establishes the potential value of the probe for facilitating investigations of HO*-mediated cellular homeostasis and injury.