Lycopene cyclase (LCY) plays a central role in branching the formation of α-type or β-type carotene. In this study, a novel lycopene cyclase (CsLcyB3) was identified from the full-length transcriptome of Crocus sativus, the major natural source of crocin with high economic value. Bioinformatic analysis revealed that CsLcyB3 had the classic features of lycopene cyclase and shared 38.85-92.39% identity with recorded functional LCYs from C. sativus. Phylogenetic analysis revealed that CsLcyB3 was grouped with LcyB-type LCYs from higher plants and algae, suggesting its function in cyclizing lycopene to form β-carotene, which was further confirmed by subsequent functional complementation assays. The physicochemical features and 3D structure were also analyzed, attempting to explain the differences in catalytic activity among different LCYs. This study confirmed the function of the novel CsLcyB3, which will expand the understanding of carotenoid biosynthesis in C. sativus and provide a new candidate for the bioengineered production of valuable carotenoids.