A new type of cascade biocatalysis was developed for one-pot enantioselective conversion of a mesoor racemic epoxide to an α-hydroxy ketone in high ee via an epoxide hydrolase-catalyzed hydrolysis of the epoxide, an alcohol dehydrogenase-catalyzed oxidation of the diol intermediate, and an enzyme-catalyzed cofactor regeneration. In vitro cascade biotransformation of meso-epoxides (cyclopentene oxide 1a, cyclohexene oxide 1b, and cycloheptene oxide 1c) was achieved with cell-free extracts containing recombinant SpEH (epoxide hydrolase from Sphingomonas sp. , BDHA (butanediol dehydrogenase from Bacillus subtilis BGSC1A1), and LDH (lactate dehydrogenase form Bacillus subtilis) or NOX (NADH oxidase from Lactobacillus brevis DSM 20054), respectively, giving the corresponding (R)-α-hydroxycyclopentanone 3a, (R)-α-hydroxycyclohexanone 3b, and (R)-α-hydroxycycloheptanone 3c in 98−99% ee and 70−50% conversion with TTN of NAD + -recycling of 5500−26 000. Cascade catalysis with mixed cells of Escherichia coli (SpEH) and E. coli (BDHA-NOX) converted 100−300 mM meso-epoxides 1a−1c to (R)-α-hydroxy ketones 3a−3c in 98−99% ee and 85−57% conversion. Cells of E. coli (SpEH-BDHA-NOX) coexpressing all three enzymes were also proven as good catalysts for the cascade conversion of 100−200 mM meso-epoxides 1a− 1c, giving (R)-α-hydroxy ketones 3a−3c in 98−99% ee and 79−52% conversion. The cascade biocatalysis for one-pot synthesis of α-hydroxy ketone in high ee was also successfully demonstrated with a racemic epoxide (1,2,3,4-tetrahydronaphthalene-1,2oxide 1d) as the substrate. By using two whole-cells based approaches, (R)-α-hydroxytetralone 3d was obtained in 99% ee and 49−40% conversion from 20 to 5 mM racemic epoxide 1d. Preparative cascade biotransformation of cyclohexene oxide 1b gave (R)-α-hydroxycyclohexanone 3b in 98% ee with 70% isolated yield. The developed new type of cascade biocatalysis is enantioselective, green, and often high yielding. The concept might be generally applicable to produce other useful enantiopure α-hydroxy ketones from the corresponding meso-or racemic epoxides by cascade catalysis using appropriate enzymes.