The invention discloses a method for preparing Globotriose and analogues of the Globotriose through a one-pot enzyme method. The method comprises the following steps: by using galactose or (2-deoxy galactose, 4-deoxy galactose and 6-deoxy galactose), lactose, UTP (uridine triphosphate), ATP (adenosine triphosphate), dithiothreitol, MgCl2, yeast inorganic pyrophosphatase and a Tris-HCl buffer solution as a reaction system, respectively adding galactokinase SpGalK, UTP-glucose-1-uridine phosphate invertase SpGalU and alpha-(1->4) galactosyl transferase LgtC for reaction; performing centrifugation and deproteinization, collecting a supernatant containing the Globotriose, and purifying through an activated carbon chromatographic column and a molecular sieve column, and performing freezing and drying to obtain the powdery Globotriose and the analogues thereof. According to the method, a main substrate is the galactose which is cheap and wide in source; the adopted enzymes SpGalK, SpGalU and LgtC are wide in substrate adaptability, and possibility is provided for mass quick production of the Globotriose and the analogues of the Globotriose.