7β-Hydroxysteroid dehydrogenase (7β-HSDH) catalyzes the reversible reaction between 7-ketolithocholic acid (7K-LCA) and ursodeoxycholic acid (UDCA). However, its much lower forward reaction activity led to the unsatisfactory UDCA production. Here, by autodocking 7K-LCA and UDCA into the structure of Hyphomicrobium sp. 7β-hydroxysteroid dehydrogenase (Hs7β-HSDH) respectively, several key amino acids in the substrate/product channel were identified for virtual mutagenesis. After three-round screening, a dominant mutant F152L/W101N was obtained, which increased forward reaction activity by 3.2-fold and decreased reverse reaction activity by 3.6-fold under optimal conditions: pH 7.5 and 30 °C. Compared to the wild-type, the mutant significantly improved the binding affinity (K