Polydatin, a glycosylated derivative of resveratrol, has better structural stability and biological activity than resveratrol. Polydatin is the extract of Polygonum cuspidatum, which has various pharmacological effects. Owing to its Crabtree-negative characteristics and high supply of malonyl-CoA, Yarrowia lipolytica was selected to produce polydatin. Initially, the resveratrol synthetic pathway was established in Y. lipolytica. By enhancing the shikimate pathway flow, redirecting carbon metabolism, and increasing the copies of key genes, a resveratrol yield of 487.77 mg/L was obtained. In addition, by blocking the degradation of polydatin, its accumulation was successfully achieved. Finally, by optimizing the glucose concentration and supplementing with two nutritional marker genes, a high polydatin yield of 6.88 g/L was obtained in Y. lipolytica, which is the highest titer of polydatin produced in a microbial host to date. Overall, this study demonstrates that Y. lipolytica has great potential for glycoside synthesis.