In this study, the flavonoid3′ hydroxylase (F3′H) gene was cloned from Chrysanthemum morifolium color petal mutant CQ17mu by RTPCR. The fulllength open reading frame of the gene was 1 527 bp, encoding 508 amino acids, which was 99% similarity with the known C. morifolium CmF3′H, so it was named CmF3′Ha. Amino acid sequence analysis showed that the protein encoded by CmF3′Ha had a conserved F3′H domain and belonged to P450 super family. Multiple sequence alignment and phylogenetic analysis showed that CmF3′Ha was closely related to F3′H of other Chrysanthemum. Real time PCR analysis showed that the expression level of CmF3′Ha gene in CQ17mu petals was significantly higher than that in CQ17 pink petals, indicating that CmF3′Ha was involved in anthocyanin metabolism in CQ17mu petals. Furthermore, a 1 086 bp promoter sequence of F3′H gene upstream of CQ17mu was obtained by chromosome walking method. Analysis showed that the sequence contained not only core promoter elements such as TATAbox, but also multiple MYB and MYC binding sites, multiple light response elements and hormone response elements. This study proved that the CmF3′Ha gene of C. morifolium was positively correlated with plant anthocyanin accumulation and participated in the anthocyanin synthesis process of color stripe, which provided a theoretical reference for the molecular breeding of C. morifolium.