The invention discloses a preparation method of galactose-N-disaccharide. The preparation method comprises the steps of adding glucose raw materials, phosphorylase, UDP-glucose hexose phosphate uranyl transferase, lactose-N-disaccharide phosphorylase, UDP-glucose epimerase, N-acetylgalactosamine and a phosphate buffer solution into a same reaction system to generate a common effect so as to synthesize a target product, namely galactose-N-disaccharide. The preparation method is simple, low in preparation cost and applicable to wide popularization and application, the used raw materials are easily available, and conditions are mild.