Developing a single nanosensor capable of detecting multiple metal ions or biomolecules remains a challenge. Here, we successfully developed a sensing platform for the efficient detection of various metal ions with orange-emissive sulfur-doped organosilica nanodots (S-OSiNDs). The S-OSiNDs were prepared via a one-pot solvothermal treatment of urea, citric acid, and bis[3-(triethoxysilyl)propyl]tetrasulfide in N,N-dimethylformamide. The as-prepared S-OSiNDs showed a turn-off fluorescence response toward multiple metal ions including Cu2+, Fe3+, [PdCl4]2–, Ag+, Hg2+, and Bi3+, realizing the rapid and sensitive detection with a very low detection limit of 0.6 nM (for Cu2+). In addition, the metal ion-induced fluorescence quenching of S-OSiNDs could be selectively restored by glutathione (GSH), exhibiting a sensitive GSH detection capability with low detection limits ranging from 0.03 to 0.2 μM. On the basis of the metal ion/GSH-triggered on–off–on regulation of the S-OSiNDs’ fluorescence, we successfully realized the detection of Cu2+, Fe3+, [PdCl4]2–, Ag+, Hg2+, and Bi3+ and achieved cancer/normal cell identification via fluorescence microscopic imaging. Overall, the S-OSiNDs may possess great potential for the detection of multiple metal ions in environmental monitoring and clinical diagnosis, and may also serve as a robust platform for cancer cell imaging and identification because of their capacity of highly sensitive sensing of GSH, which is overexpressed in many cancer cells. Furthermore, the present work also demonstrates that S-OSiNDs can be used for the facile synthesis of metal-incorporated nanoparticles, which we believe may find various applications in the future.