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Ultraviolet-ozone (UV-O₃) treatment is a simple but effective technique for surface cleaning, surface sterilization, doping, and oxidation, and is applicable to a wide range of materials. In this study, we investigated how UV-O₃ treatment affects the optical and electrical properties of molybdenum disulfide (MoS₂), with and without the presence of a dielectric substrate. We performed detailed photoluminescence (PL) measurements on 1–7 layers of MoS₂ with up to 8 min of UV-O₃ exposure. Density functional theory (DFT) calculations were carried out to provide insight into oxygen-MoS₂ interaction mechanisms. Our results showed that the influence of UV-O₃ treatment on PL depends on whether the substrate is present, as well as the number of layers. Additionally, 4 min of UV-O₃ treatment was found to be optimal to produce p-type MoS₂, while maintaining above 80% of the PL intensity and the emission wavelength, compared to pristine flakes (intrinsically n-type). UV-O₃ treatment for more than 6 min not only caused a reduction in the electron density but also deteriorated the hole-dominated transport. It is revealed that the substrate plays a critical role in the manipulation of the electrical and optical properties of MoS₂, which should be considered in future device fabrication and applications.