Transition metal dichalcogenide‐based quantum dots are promising materials for appli‐ cations 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 photo‐ luminescence. The synthesized m‐MoS2‐QDs showed clear photophysical characteristic peaks de‐ rived 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 be‐ cause 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