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Flavonoids are important bioactive components in Dendrobium officinale, a medicinal orchid. They are involved in many biological activities, including protecting plants against biotic and abiotic stresses. Research on the key genes related to flavonoid biosynthesis in D. officinale is limited. In this study, one of the key flavonoid biosynthesis genes, flavanone 3-hydroxylase (F3H), was characterized from D. officinale. The open reading frame of DoF3H was 1 134 bp long and it encoded a 377-amino acid protein. The DoF3H protein showed considerably high homology with F3H proteins from other plant species and shared a common evolutionary ancestor with other F3Hs. DoF3H transcripts were detected in different organs of adult plants and mainly accumulated in flowers, followed by roots, stems and leaves, a pattern that was similar to the content of flavonoids. Recombinant DoF3H protein, which was localized in the cytosol, could convert naringenin to dihydrokaempferol. The mRNA levels of DoF3H were significantly induced by salt and cold stresses. Furthermore, the heterologous expression of DoF3H in Escherichia coli conferred it higher tolerance to salt and cold stresses. These results provide insight into the molecular function of DoF3H in the biosynthesis of flavonoids, and provide a new application for improvement of abiotic tolerance in D. officinale. [ABSTRACT FROM AUTHOR]