Lacto-N-difucohexaose II (LNDFH II), one of the oligosaccharides in human milk, received attention because of its importance in infant health. We herein report an efficiently engineered strain for the biosynthesis of LNDFH II using systematic design and metabolic engineering in Escherichia coli BL21(DE3). The engineered strain was constructed based on the constructed lacto-N-tetraose (LNT)-producing strain by the additional expression of a recombinant GDP-fucose de novo pathway and a bacterial α-1,4-fucosyltransferase. Then, the LNDFH II synthesis pathway was divided into two modules and fine-tuned at transcriptional level, remarkably improving the LNDFH II production. Furthermore, the strengthening of the NADPH regeneration pathway helped with LNDFH II synthesis. In addition, the wcaJ gene, associated with the UDP-fucose bypass pathway, was silenced to improve further LNDFH II production in shake-flask cultivation (0.93 g/L). The final fed-batch cultivation of the engineered strain produced 9.02 g/L of LNDFH II. This study provided a strategy for the effectively producing of complex and branched human milk oligosaccharides in E. coli.