Abstract: 【Objective】 To clone the flavonol synthase （FLS） genes from Dendrobium devonianum （DdFLSs） and analyze their in vitro functions， which could provide theoretical basis for further exploring the regulatory mechanism of flavonoid biosynthesis in Dendrobium devonianum.【Method】 Based on the transcriptome sequencing data of Dendrobium devonianum leaves， candidate DdFLSs genes were screened and cloned. Bioinformatics analysis and prokaryotic system expression were performed on the cloned DdFLS genes， and the catalytic activity of DdFLSs was verified by in vitro enzyme activity assay.【Result】 Two DdFLSs genes were successfully cloned and named DdFLS1 and DdFLS2 respectively. The open reading frames （ORF） of DdFLS1 and DdFLS2 genes were both 1002 bp in length， each encoding 333 amino acids， with relative molecular weights of 37.54 kD and 37.71 kD respectively. Both proteins were unstable hydrophilic proteins and contained typical conserved domains including 2OG-Fe（II）-Oxy， PcbC， and PLN02704， belonging to the Fe²⁺ and 2-oxoglutarate-dependent dioxygenase （2-ODD） superfamily. For the secondary structure of both proteins， α-helix and random coil accounted for a high proportion， and their tertiary structures shared 70% identity with the template B0FYE8.1.A Flavonol synthase. The amino acid sequence similarity of DdFLS1 and DdFLS2 with FLS proteins from Arabidopsis thaliana， Allium cepa and Dendrobium officinale ranged from 75.98% to 98.70%. Among them， DdFLS1 and DdFLS2 had the highest amino acid sequence similarity with FLS from Dendrobium officinale， which were in the same genus， and clustered in the same small branch in the phylogenetic tree， indicating the closest genetic relationship between them. Under the induction condition of 0.5 mmol/L isopropyl β-D-thiogalactoside （IPTG） at 16 ℃ for 14 h， DdFLS1 and DdFLS2 proteins were successfully expressed in the prokaryotic expression system. The purification effect was the best when using 250 mmol/L imidazole elution buffer. In vitro enzyme activity verification showed that， DdFLS1 and DdFLS2 proteins did not exhibit typical FLS activity， that was， they could not catalyze dihydrokaempferol， dihydroquercetin， and dihydromyricetin to generate corresponding flavonols. However， they could catalyze naringenin to generate dihydrokaempferol， indicating that they possessed the activity of flavanone 3-hydroxylase （F3H）.【Conclusion】 Two DdFLSs genes have been cloned from Dendrobium devonianum， their encoded proteins do not have FLS activity but have F3H activity. This may be due to mutations in two key sites of DdFLS1 and DdFLS2 proteins， leading to the transformation of their activities.