To verify the correlation between the Salvia miltiorrhiza F3'5'H (SmF3'5'H) and the phenotype of S.miltiorrhiza flower.This study cloned and analyzed the F3'5'H genes from S.miltiorrhiza 99-3 with purple flowers and some S.miltiorrhiza plants with white flowers.Methods:The total RNA was extracted from S.miltiorrhiza with purple flowers and some S.miltiorrhiza with white flowers.And it was reversed and transcribed into cDNA.Using this cDNA as a template, the full-length of F3'5'H gene order was obtained by PCR method.Bioinformatics analysis was used to analyze the characteristics of physicochemical properties, domains, system evolution and other characteristics.The subcellular localization and transmembrane region etc.of the protein were also predicted.The gene expression features of the genes were detected by real-time quantitative PCR.The transgenic hairy roots of genes were obtained by hairy root genetic transformation method.Results:The length of F3'5'H was 1551 bp, encoding 516 amino acids, and the relative molecular mass was 57.4 kDa.This gene is highly expressed in the flowers of S.miltiorrhiza.One SNP was found in 42 samples (25 purple flowers; 17 white flowers) of S.miltiorrhiza plants.This locus showed a stable change related to flower color in S.miltiorrhiza with purple and white flowers.Phylogenetic analysis showed that F3'5'H had high sequence homology with the F3'5'H of Glandularia x hybrida.The hairy roots over-expressing F3'5'H were obtained.Conclusion:This study cloned and analyzed the F3'5'H from S.miltiorrhiza.These results lay the foundation for further research on the F3'5' H function of Salvia miltiorrhiza .