<p>Investigating the roles of oxygen vacancy in two-dimensional (2D) semiconductors is crucial for optimizing its device performance and enabling future applications. The as-cleaved 2D Ga₂O₃ has two different coordinated oxygen sites (OI and OII), however previous works focus only on the OI vacancy site. In this work, the structural, electronic, and electron mobility properties of oxygen-deficient 2D Ga₂O₃ are systematically elucidated through first-principles calculations with Perdew-Burke-Ernzerhof (PBE) and Heyd-Scuseria-Ernzerh (HSE) functionals, as well as the deformation potential (DP) theory. The formation energy calculations illustrate that the oxygen vacancy is energetically favorable at the OII site (Ga₂O₃V_OII) with well structural stabilities. Accompanying with oxygen vacancy formation, an obvious mid-gap state is generated and serves as the deep donor state, which decreases the bandgap of Ga₂O₃V_OII more than 30% to 1.60&#xa0;eV using PBE functional (or 3.26&#xa0;eV under HSE), leading to the red-shift of the optical absorption edge. The bandgaps vary from 1.78 to 0.65&#xa0;eV under biaxial strain modulations from −8% compressive to 8% tensile, which is elaborated by the band edge variations of conduction band maximum (CBM) and valence band minimum (VBM) with respect to the vacuum levels. The unstrained Ga₂O₃V_OII possesses the electron mobilities of 2044.76 and 2512.74 cm²V⁻¹s⁻¹ along x and y directions, which are increased 6 times and 18 times higher under 2% tensile and −4% compressive strains, respectively. Moreover, a considerably high anisotropy ratio of ~ 46 is achieved under −4% compressive strain. The outstanding stabilities, strain-tunable bandgap, exceptional electron mobility and strong anisotropy ratio observed in oxygen-deficient 2D Ga₂O₃ hold promising for applications as nanoscale optoelectronic devices, particularly for Ga₂O₃V_OII film grown on SiC substrate.</p>