Human milk oligosaccharides (HMOs) are bioactive oligosaccharides with characterization of complexity and indigestion, which support the progress of immune system, gut microenvironment, and brain in infants. In this work, 3-fucosyllactose (3-FL) was successfully synthesized in recombinant plasmid-free Escherichia coli by strengthening GDP-l-fucose pathway. For this, the native promoters of manB, manC, gmd, and wcaG in chromosome of E. coli were replaced by PJ23119, a constitutive promoter. To further increase the 3-FL production, multiple copies of fut3Bc (encoding α1,3-fucosyltransferase from Neobacillus cucumis) were integrated into the genome of engineered strains to replace the native genes arsB, ldhA, poxB, and manXYZ, respectively. The titer of 3-FL was boosted to 3.17 g/L via overexpressing manA with promoter PJ23119 in chromosome of the engineered hosts. Ultimately, in a 5 L bioreactor, the titer of 3-FL achieved 28.13 g/L by fed-batch culture at 60 h, with a productivity of 0.54 g/L/h and a yield of 0.95 mol/mol lactose, without using plasmids and antibiotics. Importantly, only 3-FL could be synthesized in fut3Bc-based strains without producing other HMOs, showing great potential in either the purity or productivity.