The invention discloses a construction method and application of a high-yield lactoyl-N-tetrasaccharide microorganism, and belongs to the field of microbial genetic engineering. According to the method, a strain which is constructed in an early stage and is used for efficiently producing a precursor substance lactoyl-N-trisaccharide II is used as an original strain, and a key gene for synthesizing lactoyl-N-tetrasaccharide is over-expressed, so that the strain has the synthesis capability of producing lactoyl-N-tetrasaccharide. An efficient beta-1, 3-galactosyl transferase gene is screened, the coexpression beta-1, 3-galactosyl transferase gene and a UDP-glucose 4 epimerase gene (galE) which is a key gene for strengthening a UDP-galactose pathway are reasonably designed on a carrier pCDFDuet-1, so that the synthesis of the lactoyl-N-tetrasaccharide is improved, and in a shake flask experiment, the yield of the lactoyl-N-tetrasaccharide is increased, and the yield of the lactoyl-N-tetrasaccharide is increased. In a bottle shaking experiment, the capacity of producing the lactoyl-N-tetrasaccharide by the escherichia coli is 3.04 g/L, the yield of the lactoyl-N-tetrasaccharide in a 3L fermentation tank reaches 25.49 g/L, and the construction method has an industrial application prospect.