Fraxetin, which is a simple coumarin, is a phytochemical present in medicinal plants, such as Fraxinus rhynchophylla, and Cortex Fraxini. In plants, it serves as a controller of iron homeostasis. The health-enhancing activities of fraxetin, such as anticancer, neuroprotective and antibacterial activities, are known. Scopoletin 8-hydroxylase (S8H) is a key enzyme involved in the synthesis of fraxetin from scopoletin. Scopoletin can be synthesized either from esculetin by O-methylation or from ferulic acid by feruloyl CoA 6′-hydroxylase (F6′H) and 4-coumaric acid CoA ligase (4CL). To enable fraxetin synthesis, the fraxetin biosynthesis pathway was introduced into Escherichia coli. Three distinct routes, from ferulic acid, esculetin, and scopoletin, were designed for the synthesis of fraxetin. In the first approach, E. coli strain harboring S8H was used and found to synthesize 84.8 μM fraxetin from 100 μM scopoletin. Two E. coli strains were used for the other two approaches because these approaches required at least two enzymatic reactions. Through this approach, 41.4 μM fraxetin was synthesized from 100 μM esculetin, while 33.3 μM fraxetin was synthesized from 100 μM ferulic acid.