Ursodeoxycholic acid (UDCA) can be used as a drug to treat various liver and bile diseases. Currently, the biological synthesis of UDCA is predominantly conducted via a two-step enzymatic process in which synthesis is catalyzed by 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) in succession, utilizing chenodeoxycholic acid (CDCA) as the substrate. In this study, an engineered