Ag₂S quantum dots (QDs) emitting in the second near-infrared region (NIR-II, 1.0–1.4 μm) are demonstrated as a promising fluorescent probe with both bright photoluminescence and high biocompatibility for the first time. Highly selective in vitro targeting and imaging of different cell lines are achieved using biocompatible NIR-II Ag₂S QDs with different targeting ligands. The cytotoxicity study illustrates the Ag₂S QDs with negligible effects in altering cell proliferation, triggering apoptosis and necrosis, generating reactive oxygen species, and causing DNA damage. Our results have opened up the possibilities of using these biocompatible Ag₂S QDs for in vivo anatomical imaging and early stage tumor diagnosis with deep tissue penetration, high sensitivity, and elevated spatial and temporal resolution owing to their high emission efficiency in the unique NIR-II imaging window.