AbstractCordycepin (COR) has attracted extensive attention for its medical value. Currently, separation and extraction from Cordyceps militaris is the main way to obtain COR, but it is time‐consuming and low efficiency. Heterologous synthesis of COR holds great promise for its industrial production. In this work, five COR synthases of different sources were obtained by homology screening, and were integrated into the genome of BY4741. Fermentation and determination of COR found that EA1 and EA2 from Emericellopsis atlantica were more suitable for COR synthesis. Then, ADO1 gene encoding adenosine kinase was knocked down, ADE4 gene encoding amidophosphoribosyltransferase and truncated cpdBN gene encoding 2′,3′‐cyclic‐nucleotide 2′‐phosphodiesterase/3′‐nucleotidase were overexpressed to enhance the accumulation of COR. Finally, the COR titer reached 725.16 mg L−1 in 5‐L bioreactor. This study lays a foundation for the heterologous synthesis and industrial production of COR, and also provides a reference for heterologous synthesis of other high value‐added chemicals.