Low toxicity and fluorescent nanomaterials have many advantages in biological imaging. Herein, a novel and facile aqueous-phase approach to prepare biocompatible and fluorescent Ag2S nanoclusters (NCs) is designed and investigated. The resultant Ag2S NCs show tunable luminescence from the visible red (624 nm) to the near infrared (NIR; 724 nm) corresponding to the increasing size of the NCs. The key for preparing tunable fluorescent Ag2S NCs is the proper choice of capping reagent, glutathione (GSH), and the novel sulfur-hydrazine hydrate complex as the S2− source. As a naturally occurring and readily available tripeptide, GSH functions as an important scaffold to prevent NCs from growing large nanoparticles. Additionally, GSH is a small biomolecule with several functional groups, including carboxyl and amino groups, which suggests the resultant Ag2S NCs are well-dispersed in aqueous solution. These advantages make the as-prepared Ag2S NCs potentially applicable to biological labeling as well. For example, the resultant Ag2S NCs are used as a probe for MC3T3-EI cellular imaging.