3-Fucosyllactose (3-FL) is a typical human milk oligosaccharides (HMOs) and plays a significant physiological function in the growth and brain development of infants. In this study, efficient biosynthesis of 3-FL was achieved by metabolic engineering of Escherichia coli BL21star(DE3). The α-1,3-fucosyltransferase gene futA from Helicobacter pylori and the L-fucokinase/GDP-L-fucose pyrophosphorylase gene fkp from Bacteroides fragilis were co-introduced into the initial strain, E. coli BL21star(DE3)ΔlacZΔwcaJ, resulting in the production of 1.01 g/L of 3-FL in a shake flask. By using the CRISPR/Cas9 system to delete the genes related to the fucose metabolism pathway, including the L-fucose isomerase gene fucI, the L-fucose kinase gene fucK and the L-rhamnose isomerase gene rhaA, the yield of 3-FL increased to 1.36 g/L. The α-1,3-fucosyltransferase FT1 from H. pylori NCTC11637 was the most effective in 3-FL biosynthesis, and the recombinant strain BLWFA-T expressing FT1 produced 1.56 g/L of 3-FL. Subsequently, the expression level of the fucose metabolism pathway-related genes was optimized, and the recombinant strain BLWFR-T2 expressing fkp through plasmid pETDuet-1 and expressing FT1 through plasmid pRSFDuet-1 produced 2.58 g/L of 3-FL. Finally, the regeneration of the cofactor guanosine triphosphate (GTP) was enhanced, and the recombinant strain BLWFR-T7 overexpressing the guanylate kinase gene gmk and the inosine-guanosine kinase gene gsk produced the highest level of 3-FL (3.01 g/L). After fed-batch fermentation for 78 h, BLWFA-T7 produced the highest yield of 3-FL (27.82 g/L) in a 5 L fermentor. This study provides a research basis for efficient microbial synthesis of 3-FL and other HMOs.