Lutein　is　a　high-value　carotenoid　with　many　human　health　benefits.　Lycopene　β-　and　ε-cyclases　(LCYB　and　LCYE,　respectively)　catalyze　the　cyclization　of　lycopene　into　distinct　downstream　branches,　one　of　which　is　the　lutein　biosynthesis　pathway,　via　α-carotene.　Hence,　LCYB　and　LCYE　are　key　enzymes　in　lutein　biosynthesis.　In　this　study,　the　coding　genes　of　two　lycopene　cyclases　(CsLCYB　and　CsLCYE)　of　a　lutein-enriched　marine　green　microalga,　Chlorella　sorokiniana　FZU60,　were　isolated　and　identified.　A　sequence　analysis　and　computational　modeling　of　CsLCYB　and　CsLCYE　were　performed　using　bioinformatics　to　identify　the　key　structural　domains.　Further,　a　phylogenetic　analysis　revealed　that　CsLCYB　and　CsLCYE　were　homogeneous　to　the　proteins　of　other　green　microalgae.　Subcellular　localization　tests　in　Nicotiana　benthamiana　showed　that　CsLCYB　and　CsLCYE　localized　in　chloroplasts.　A　pigment　complementation　assay　in　Escherichia　coli　revealed　that　CsLCYB　could　efficiently　β-cyclize　both　ends　of　lycopene　to　produce　β-carotene.　On　the　other　hand,　CsLCYE　possessed　a　strong　ε-monocyclase　activity　for　the　production　of　δ-carotene　and　a　weak　ε-bicyclic　activity　for　the　production　of　ε-carotene.　In　addition,　CsLCYE　was　able　to　catalyze　lycopene　into　β-monocyclic　γ-carotene　and　ultimately　produced　α-carotene　with　a　β-ring　and　an　ε-ring　via　γ-carotene　or　δ-carotene.　Moreover,　the　co-expression　of　CsLCYB　and　CsLCYE　in　E.　coli　revealed　that　α-carotene　was　a　major　product,　which　might　lead　to　the　production　of　a　high　level　of　lutein　in　C.　sorokiniana　FZU60.　The　findings　provide　a　theoretical　foundation　for　performing　metabolic　engineering　to　improve　lutein　biosynthesis　and　accumulation　in　C.　sorokiniana　FZU60.　©　2023　by　the　authors.