Cholesterol oxidase, a flavoenzyme, catalyzes two reactions in one active site: oxidation and isomerization. This enzyme has been isolated from a variety of microorganisms, mostly from actinomycetes. This enzyme has been widely used in clinical laboratories for cholesterol assays and was subsequently determined to have other potential applications. Engineering of cholesterol oxidase have enabled the identification of critical residues, and the information derived could lead to the rational development of improved types of the enzyme with increased stability and better functional properties. This review is the first that exclusively summarizes the reported results on the engineering of bacterial cholesterol oxidases aimed at improving their thermal and chemical stability, catalytic activity, and substrate specificity.