Ene-reductases (ERs) from the old yellow enzyme (OYE) family are flavin-dependent enzymes that catalyze the asymmetric reduction of electronically activated carbon–carbon double bonds. These enzymes have attracted increased attention from industry as the enzymatic reduction usually proceeds with enhanced chemoselectivity and elevated stereoselectivity. Although other enzyme families are capable of catalyzing the reduction of alkene moieties, the large majority of the biocatalytic applications involve members of the OYE family. Therefore this book chapter focuses on the recent biosynthetic application of OYEs for the conversion of a wide range of substrates, encompassing α,β-unsaturated aldehydes (enals), α,β-unsaturated ketones (enones), α- or β-substituted nitroalkenes, α,β-unsaturated esters, and α,β-unsaturated nitriles. Furthermore, OYEs have recently been used in creating novel chemoenzymatic as well as enzymatic one-pot multistep processes, particularly in combination with alcohol dehydrogenases (ADHs). Selections of the most relevant examples are presented here. A brief overview regarding the recent trend of replacing the nicotinamide phosphate coenzymes with less expensive and chemically synthesized analogues is also described. Finally, techniques of protein engineering aimed at improving the productivity, stability, and selectivity of OYEs for industrial applications are discussed.