The invention relates to a preparation method of uridine diphosphate glucuronic acid. Uridine diphosphate glucose is oxidized by uridine diphosphate glucose dehydrogenase to generate uridine diphosphate glucuronic acid, and lactic dehydrogenase is added for double-enzyme coupling reaction, so that cyclic regeneration of NAD<+> is realized, the addition of NAD<+> is reduced, the cost is saved, and at the same time, the circulation of NAD<+> / NADH reduces the feedback inhibition effect of the byproduct NADH on the product uridine diphosphate glucuronic acid, and the generation amount of the target product can reach 1.01 mg / mL. The nucleotide sequence of lactic dehydrogenase is optimized and modified, so that the target protein can be better expressed in escherichia coli, and efficient synthesis of the target product uridine diphosphate glucuronic acid is facilitated. In-vitro reaction is adopted, a reaction system is more controllable, and compared with intracellular reaction, in-vitro reaction can be more beneficial to reaction under limited conditions.