Abstract 2′-Fucosyllactose (2′-FL) is the most abundant human milk oligosaccharide (HMO), playing vital roles in promoting infant gut health, enhancing immunity, and defending against pathogens. However, conventional 2′-FL biosynthesis typically relies on exogenous supplementation of lactose or fucose precursors, leading to high costs and process complexity. In this study, we report a streamlined de novo biosynthesis process for 2′-FL using glucose as the sole carbon source, achieved through systematic engineering of endogenous precursor pathways in Escherichia coli . First, the complete biosynthetic route from glucose to lactose and then to 2′-FL was reconstructed in a chassis strain capable of producing fucose. Unlike previous studies that primarily focused on the GDP-fucose supply module, this work systematically balanced the metabolic flux between phosphorylated and non-phosphorylated glucose pools to coordinate the supply of precursors for both lactose and GDP-fucose synthesis. This balance was achieved by reconstructing the glucose uptake system, modulating the expression of key enzymes at critical metabolic nodes, and eliminating competing pathways. Subsequently, the endogenous lactose synthesis pathway was further enhanced through coordinated overexpression of pgm , galU , and galE , and the pentose phosphate pathway and purine salvage pathway were optimized to enhance the supply of NADPH and GTP. Using this strategy, an engineered E. coli strain for efficient 2′-FL production was successfully constructed. The engineered strain produced 7.11 g/L 2′-FL in shake-flask fermentation and achieved a titer of 43.2 g/L in a 3-L bioreactor after 58 h, representing the highest reported yield for de novo 2′-FL synthesis from glucose as the sole carbon source. This work provides a promising and simplified strategy for the cost-effective industrial production of 2′-FL.