7-Ketolithocholic acid (7-KLCA) is an important intermediate for the synthesis of ursodeoxycholic acid (UDCA). UDCA is the main effective component of bear bile powder that is used in traditional Chinese medicine for the treatment of human cholesterol gallstones. 7-Hydroxysteroid dehydrogenase (7-HSDH) is the key enzyme used in the industrial production of 7-KLCA. Unfortunately, the natural 7-HSDHs reported have difficulty meeting the requirements of industrial application, due to their poor activities and strong substrate inhibition. In this study, a directed evolution strategy combined with high-throughput screening was applied to improve the catalytic efficiency and tolerance of high substrate concentrations of NADP+-dependent 7-HSDH from Clostridium absonum. Compared with the wild type, the best mutant (7-3) showed 5.5-fold higher specific activity and exhibited 10-fold higher and 14-fold higher catalytic efficiency toward chenodeoxycholic acid (CDCA) and NADP+, respectively. Moreover, 7-3 also displayed significantly enhanced tolerance in the presence of high concentrations of substrate compared to the wild type. Owing to its improved catalytic efficiency and enhanced substrate tolerance, 7-3 could efficiently biosynthesize 7-KLCA with a substrate loading of 100mM, resulting in 99% yield of 7-KLCA at 2h, in contrast to only 85% yield of 7-KLCA achieved for the wild type at 16h.