2ʹ-fucosyllactose (2ʹ-FL), a major component of human milk oligosaccharides, plays important roles in immune regulation and brain development in humans. The probiotic gut bacterium Escherichia coli Nissle 1917 is a highly promising synthetic biology chassis strain for microbial production of 2′-FL. To further expand its application potential, a complete biosynthetic pathway for 2ʹ-FL was constructed in Nissle 1917. The expression of endogenous pathway genes was enhanced by replacing promoters and ribosome-binding sites (RBSs), while the supply of the precursor GDP-L-fucose was increased by suppressing branch and competing pathways. To address the low heterologous expression efficiency of α-1,2-fucosyltransferase, a directed evolution strategy based on multiple sequence alignment was applied to improve the solubility and catalytic efficiency of wcfB. Application of these combined metabolic engineering strategies enabled the engineered E. coli Nissle 1917 strain to achieve 2ʹ-FL titers of 9.92 g•L-1 in shake flasks and 58 g•L-1 in a 1.5 L bioreactor. This study successfully established E. coli Nissle 1917 as a microbial chassis for 2ʹ-FL production and expanded its library of molecular biology operational elements, thereby providing new insights into advancing fundamental research and biotechnological applications of this strain in synthetic biology. Moreover, engineering the probiotic E. coli Nissle 1917 for 2′-FL production through synthetic biology provides valuable insights for integrating next-generation probiotics with synthetic biology.