To overcome economic challenges in microalgal biofuel production, this study investigates the overexpression of peroxisome-localized peroxisomal biogenesis factor 11 (PEX11) to enhance lipid biosynthesis and improve salt stress resistance in Tetradesmus obliquus, aiming to advance microalgal biofuel production. Transgenic strains PEX11-2-1 and PEX11-2-2 exhibited a 2.13- and 2.51-fold increase in neutral lipid content and more cellular lipid droplets compared to WT, along with lipid yield and biomass escalating to 255.45 and 815.15 mg/L, respectively. This enhancement resulted from the redistribution of carbon precursors, increased intracellular reactive oxygen species, enhanced NADPH synthesis, and upregulation of lipid synthesis genes. Additionally, PEX11 improved salt stress tolerance by upregulating the expression of stress-responsive genes, including SnRK2 and PYRC. Fatty acid profile alterations, with increases in saturated fatty acids C16:0 and monounsaturated fatty acids C18:1, and decreases in polyunsaturated fatty acids, facilitated high-quality biofuel production. These findings highlight novel insights for advancing microalgae-based biorefinery.