As one key component of human milk oligosaccharides, lacto-N-neotetraose (LNnT) plays an important role in the growth and development of infants. In order to explore the efficient method for biosynthetic of LNnT and to investigate the influence of module optimization on LNnT synthesis of Escherichia coli, E. coli BL21(DE3)ΔlacZ was used as the initial strain. And the synthetic pathway of LNnT was divided into the following three modules based on the key metabolites in synthetic pathway, module A for the exogenous enzymatic pathway, module B for the synthetic pathway of UDP-galactose, and module C for the synthetic pathway of UDP-N-acetylglucosamine. After preliminarily optimizing the expressions of modules A, B, and C via the plasmids with different copy number, the E. coli BL21(DE3)ΔlacZ harboring the recombinant plasmids pRSF-lgtA-A.act and pET-galE produced the highest LNnT titer of 0.87g/L. Modules A and B were enhanced owing to knocking setA and ugd via CRISPR/Cas9 technique, and the titer of LNnT produced by the recombinant strain E20 was up to 1.16g/L. After optimizing the fermentation conditions of strain E20, the titer of LNnT in shake-flask cultivation was up to 1.28g/L and further was up to 15.53g/L by fed-batch fermentation in a 5L bioreactor. The highest productivity of LNnT was up to 0.43g/(L·h) during fermentation. The enhancement of LNnT synthesis in E. coli with module optimization was expected to provide theoretical basis for efficient biosynthesis of human milk oligosaccharides and to drive innovation in food industry of infant formula.