To reduce the premature release of encapsulated drug, glutathione-responsive fluorescent porous silica (pSiO 2 ) nanocarriers were developed by encapsulating Ag nanoclusters (Ag NCs) and capped with Ag nanoparticles (Ag NPs). Ag NCs/porous silica (Ag NCs/pSiO 2 ) nanospheres (NSs) have an average diameter of 45 nm and large specific surface with Brunauer-Emmett-Teller (BET) area of 453 m 2 g −1 . After loading N -(2-Mercaptopropionyl) glycine (MPG), the absorbed Ag + ions were in situ reduced to Ag NPs and capped on the outer surfaces of Ag NCs/pSiO 2 NSs as gatekeepers to regulate the release of drugs. In the absence of glutathione (GSH), the release rate is below 8% within 12 h; while in the presence of 2 mM GSH, the amount released reaches 70% within 8 h. The Ag NPs-gated Ag NCs/pSiO 2 NSs displayed excellent GSH-responsive release. The incorporated Ag NCs presented novel drug-dependent fluorescence, which could be used to trace the drug release. In addition, the Ag NPs-gated Ag NCs/pSiO 2 NSs displayed excellent antibacterial activity against both Gram-positive and Gram-negative bacteria.