In this study, a LCYB gene was cloned from Hibiscus esculentus L. by RT-PCR and RACE technique. The full-length cDNA sequence of LCYB was 1797 bp, which contained a 1509 bp open reading frame(ORF)that encoded 503 amino acids,with a predicted molecular weight (mol.wt.) of 56.288 kD and a hypothetical isoelectric point (pI) of 4.577. It shared over88% identity with the homologous proteins from Gossypium raimondii, Corchorus olitoriu. and Theobroma cacao, proving that it was highly conservative. This gene was named HyLCYB and the GeneBank accession was KX257998. Motif Scan analysis showed that HyLCYB protein had a conservative structure domain and binding domain in the position of 88-481 and 88-113 sites,respectively. Real-time PCR analysis revealed that LCYB could be expressed in different tissues of Hibiscus esculentus L., including roots, stems, leaves, flowers and fruits. The expression level of LCYB gene was the highest in mature leaves and 7 days after flowering during the process of Leaf and fruit development respectively. What’s more, an ultra-high performance liquid chromatography (UPLC) method for the determination of carotenoid contents was explored. The carotenoids in Hibiscus esculentus L. primarily consisted of lutein and β-carotene, andβ-carotene contents was also the highest in mature leaves and 7 days after flowering during the process of Leaf and fruit development respectively, which probably related to the changes in LCYB genes expression.