• LCYB from sweet osmanthus can efficiently β -cyclize lycopene to produce β-carotene. • LCYE from sweet osmanthus is able to cyclize lycopene to produce bicyclic ε-carotene. Sweet osmanthus ( Osmanthus fragrans ) flowers, as a food flavor additive, are rich in carotenoids, the majority of which are cyclized carotenoids. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the cyclization of the open ends of acyclic lycopene and manipulate the metabolic flux into different downstream branches. Although both LCYB and LCYE have been fully characterized in many plants, little information is available concerning the functions of sweet osmanthus LCYB and LCYE in carotenoid biosynthesis. In this study, we cloned two lycopene cyclase genes OfLCYB and OfLCYE in sweet osmanthus. The expression levels of OfLCYB in the petals gradually increased during flower opening, having a significant positive correlation with total carotenoid contents in each sweet osmanthus cultivar. Pigment complementation assay in Escherichia coli demonstrated that OfLCYB could efficiently β-cyclize both ends of lycopene to produce β-carotene (β,β-carotene), while OfLCYE was able to catalyze the formation of ε-carotene (ε,ε-carotene) at both ends of lycopene. The characterization of LCYB and LCYE from sweet osmanthus drives the first bifurcation step toward β-carotene and α-carotene. Our results will lay theoretical foundation for future metabolic engineering of carotenoid biosynthesis and accumulation in sweet osmanthus flowers.