AbstractStructure‐guided directed evolution of choline oxidase has been carried out by using the oxidation of hexan‐1‐ol to hexanal as the target reaction. A six‐amino‐acid variant was identified with a 20‐fold increased kcat compared to that of the wild‐type enzyme. This variant enabled the oxidation of 10 mm hexanol to hexanal in less than 24 h with 100 % conversion. Furthermore, this variant showed a marked increase in thermostability with a corresponding increase in Tm of 20 °C. Improved solvent tolerance was demonstrated with organic solvents including ethyl acetate, heptane and cyclohexane, thereby enabling improved conversions to the aldehyde by up to 30 % above conversion for the solvent‐free system. Despite the evolution of choline oxidase towards hexan‐1‐ol, this new variant also showed increased specific activities (by up to 100‐fold) for around 50 primary aliphatic, unsaturated, branched, cyclic, benzylic and halogenated alcohols.