In this study, a flavanone 3hydroxylase (F3H) gene named EmF3H (GenBank: MW767838) was cloned from Euphorbia maculata with homologous cloning, RACE technology and TailPCR. The ORF of EmF3H is 1 092 bp, encoding 364 amino acids. Bioinformatics analysis showed that the theoretical molecular weight of EmF3H protein is 40.93 kD, theoretical pI is 5.47 and EmF3H protein belongs to 2oxoglutaratede pendent dioxygenase protein family. EmF3H protein is 85.5% similar to VfF3H (ARV78456.1) and located on the independent evolutionary node in phylogenetic tree. The 1 604 bp sequence of EmF3H promoter was cloned by TailPCR and included TAATbox, CAATbox, light responsive element and so on. The analysis of realtime quantitative PCR indicated that EmF3H gene was expressed among different tissues during different growth periods, with the strongest expression in the roots at the flowering stage and the fruits at fruiting stage. These results will provide a foundation for further research on gene expression regulation of EmF3H gene and improving the biosynthesis pathway of quercetin in Euphorbia maculata.