Two-dimensional (2D) semiconductors have attracted much attention regarding their use in flexible electronic and optoelectronic devices, but the inherent poor electron mobility in conventional 2D materials severely restricts their applications. Using first-principles calculations in conjunction with Boltzmann transport theory, we systematically investigated the Si-doped 2D β-Ga2O3 structure mediated by biaxial strain, where the structural stabilities were determined by formation energy, phonon spectrum, and ab initio molecular dynamic simulation. Initially, the band gap values of Si-doped 2D β-Ga2O3 increased slightly, followed by a rapid decrease from 2.46 eV to 1.38 eV accompanied by strain modulations from −8% compressive to +8% tensile, which can be ascribed to the bigger energy elevation of the σ* anti-bonding in the conduction band minimum than that of the π bonding in the valence band maximum. Additionally, band structure calculations resolved a direct-to-indirect transition under the tensile strains. Furthermore, a significantly high electron mobility up to 4911.18 cm2 V−1 s−1 was discovered in Si-doped 2D β-Ga2O3 as the biaxial tensile strain approached 8%, which originated mainly from the decreased quantum confinement effect on the surface. The electrical conductivity was elevated with the increase in tensile strain and the enhancement of temperature from 300 K to 800 K. Our studies demonstrate the tunable electron mobilities and band structures of Si-doped 2D β-Ga2O3 using biaxial strain and shed light on its great potential in nanoscale electronics.