Rutin and quercetin, abundant in tartary buckwheat, have physiological and pharmacological functions and play roles in abiotic stress tolerance in plant. Rutin degrading enzymes (RDE) are the key enzymes for rutin metabolism. However, the RDE coding sequence information has not been available. In this study, a 1515-bp coding sequence of RDE was cloned from tartary buckwheat (named FtRDE) using 5' and 3' RACE, based on the FtRDE protein sequence. The recombinant RDE (rRDE) expressed in P.pastoris with glycosylation modification degraded rutin into quercetin and the Glu171 and Glu382 were indispensable residues for catalytic activity. FtRDE was highly expressed in seed filling stage and response to ABA and MeJA, confirmed by qRT-PCR and FtRDE promoter activity analysis in mesophyll protoplast. This study provided a new approach for the large-scale preparation of RDE by heterologous expression and production of quercetin by hydrolyzing rutin, and could be helpful for understanding the FtRDE function under stress conditions.