Cordycepin is a nucleoside antibiotic with various biological activities, which has wide applications in the area of cosmetic and medicine industries. However, the current production of cordycepin is costly and time-consuming. To construct the promising cell factory for high-level cordycepin production, firstly, the design and construction of cordycepin biosynthetic pathway were performed in Yarrowia lipolytica. Secondly, the adaptivity between cordycepin biosynthetic pathway and Y. lipolytica was enhanced by enzyme fusion and integration site engineering. Then, the production of cordycepin was improved by the enhancement of adenosine supply. Furthermore, through modular engineering, the production of cordycepin was achieved at 3588.59 mg/L from glucose. Finally, 3249.58 mg/L cordycepin with a yield of 76.46 mg/g total sugar was produced by the engineered strain from the mixtures of glucose and molasses. This research is the first report on the de novo high-level production of cordycepin in the engineered Y. lipolytica.