Silver as an extensively used antibacterial agent also poses potential threats to the environment and human health. Hence, in this work, we design a fluorescent nanoprobe by using rhodamine B isothiocyanate (RhoBS) adsorbed MoS2 nanosheets to realize sensitive and selective detection of Ag+. On the surface of RhoBS-loaded MoS2 nanosheets, Ag+ can be reduced to Ag nanoparticles, which afterward could not only lead to the detachment of RhoBS molecules and thus their recovered fluorescence but also the surface-enhanced fluorescence from RhoBS remaining adsorbed on MoS2. Such an interesting mechanism allows highly sensitive detection of Ag+ (down to 10 nM) with great selectivity among other metal ions. Moreover, we further demonstrate that our MoS2-RhoBS complex could act as a nontoxic nanoprobe to detect Ag+ in live bacteria samples. Our work resulted from an unexpected finding and suggests the promise of two-dimensional transition-metal sulfide nanosheets as a novel platform for chemical and biological sensing. © 2015 American Chemical Society.