Activatable fluorescence nanoprobes with only one kind of nanomaterial that can act as both the energy donor and acceptor simultaneously are scarce, but highly desirable for biosensing and bioimaging. In the present study, we reveal the preparation of self-quenched gold nanoclusters as a simple fluorescent turn-on probe for imaging intracellular glutathione. The self-quenched gold nanoclusters are prepared via disulfide bond-induced aggregation of gold nanoclusters. Compared with monodisperse gold nanoclusters, the developed self-quenched gold nanoclusters exhibit weak emission at 735 nm with a 40-nm red shift and much lower quantum yield (0.69%). The prepared self-quenched gold nanoclusters also possess good sensitivity and selectivity for glutathione detection, and are applicable for fluorescent turn-on imaging of intracellular glutathione.