Mandelic acid (MA) is a valuable α-hydroxy acid with applications in pharmaceuticals, cosmetics, and fine chemicals. While chemical synthesis is well established, concerns over toxicity and sustainability have driven interest in microbial production. Here, we engineered Escherichia coli for de novo MA biosynthesis by integrating enzyme screening, metabolic flux optimization, and pathway regulation. We first screened and identified an efficient hydroxymandelate synthase (HMAS) homolog from Actinosynnema mirum for MA synthesis, and subsequently enhanced the shikimate pathway along with the supply of the precursors erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP). Additionally, CRISPR interference (CRISPRi) was employed to repress competing pathways and redirect flux toward MA production. High-cell-density cultivation (HCDC) in a 5 L bioreactor demonstrated the strain’s industrial potential, achieving an MA titer of 9.58 g/L, the highest reported for microbial production. This study provides a systematic metabolic engineering approach for efficient MA biosynthesis from glucose, offering a foundation for sustainable large-scale production, demonstrating not only genetic-level optimizations, but also effective process scaling through high-cell-density cultivation, highlighting the power of pathway engineering in microbial cell factories.