Chalcone isomerase CHI is a key enzyme in the biosynthesis pathway of anthocyanin. In order to understand the synthesis mechanism of anthocyanin in O.fragrans, we detected the anthocyanin content of three O.fragrans cultivars in this study. The results showed that: (1) the anthocyanin content in ‘Chenghongdangui’ was the highest, while lower in ‘Jingui’ and ‘Zaoyingui’. (2) The fulllength cDNA sequence of OfCHI (1 069 bp) was obtained by using RACE and RTPCR methods. OfCHI encoded a deduced protein of 248 amino acids, with the relative molecular weight of 26.85 kD and isoelectric point of 6.34. (3) Multiple sequence alignment showed that O. fragrans had 68.13%, 65.86% and 63.53% similarity with Camellia nitidissima, Punica granatum and Lonicera japonica, respectively. OfCHI contained the active sites of ChI protein: Thr47, Tyr108, Met115 and Ser192. Phylogenetic tree analysis showed that OfCHI shared the same origin with other species, and had the closest relationship with Olea europaea OeCHI. (4) OfCHI expression was detected in different cultivars and tissues using qRTPCR. The results showed that OfCHI expression level was the highest in‘Chenghongdangui’, while lower in ‘Jingui’ and ‘Zaoyingui’. OfCHI expression was detected in flowers, stems and leaves of ‘Chenghongdangui’, ‘Jingui’ and ‘Zaoyingui’, and showed the same expression trend with the highest expression level in leaves. The study provides not only theoretical basis for the synthesis mechanism of anthocyanin in osmanthus, but also gene patent for developing new osmanthus cultivars of different flower color.