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Transition metal dichalcogenide-based quantum dots are promising materials for applications in diverse fields, such as sensors, electronics, catalysis, and biomedicine, because of their outstanding physicochemical properties. In this study, we propose bio-imaging characteristics through utilizing water-soluble MoS2 quantum dots (MoS2-QDs) with two different sizes (i.e., ~5 and ~10 nm). The structural and optical properties of the fabricated metallic phase MoS2-QDs (m-MoS2-QDs) were characterized by transmission electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, UV–vis absorption spectroscopy, and photoluminescence. The synthesized m-MoS2-QDs showed clear photophysical characteristic peaks derived from the quantum confinement effect and defect sites, such as oxygen functional groups. When the diameter of the synthesized m-MoS2-QD was decreased, the emission peak was blue-shifted from 436 to 486 nm under excitation by a He-Cd laser (325 nm). Density functional theory calculations confirmed that the size decrease of m-MoS2-QDs led to an increase in the bandgap because of quantum confinement effects. In addition, when incorporated into the bio-imaging of HeLa cells, m-MoS2-QDs were quite biocompatible with bright luminescence and exhibited low toxicity. Our results are commercially applicable for achieving high-performance bio-imaging probes.