Dihydroflavonol-4-reductase (DFR) is a key enzyme in phycocyanin synthesis that catalyzes the production of colorless anthocyanins from dihydroflavonols. In this experiment, petals from different organs and different developmental periods of red and white Rhododendron hybridum Hort. were used as experimental materials, and the RhDFR gene of R. hybridum Hort. was cloned with reverse transcription (RT-PCR) and RACE techniques, and the plant enzyme-linked immunoassay kit (ELISA) was used to determine petal RhDFR enzyme activity of petals at different developmental stages, and the qRT-PCR technology was used to quantify the petal RhDFR gene in different organs and different developmental stages, and the preparation and purification of RhDFR protein by pET-28a-RhDFR prokaryotic expression vector was constructed, which laid a foundation for further exploration of the function of Rhododendron DFR gene and the molecular mechanism of flower color. The results showed that: (1) successful acquisition of R. hybridum Hort. RhDFR gene is 1 253 bp long, an open reading frame of 1 035 bp, encoding 344 amino acids, and contains a NADPH-binding region and a substrate-binding region with high conservation. Phylogenetic analysis showed that R. hybridum Hort. RhDFR protein is most closely related to the DFR protein of lingonberry (Vaccinium corymbosum). (2) The analysis of ELISA kit showed that the DFR enzyme activity of petals showed in different stages of development of R. hybridum Hort. first increased and then decreased, and the DFR enzyme activity was the highest in the middle opening stage of red flower and full opening stage of white flower. (3) The qRT-PCR analysis showed that the expression of RhDFR gene in different organs was the highest in petals, and the lowest in stamens; In petals at different developmental stages of development, red flowers had higher RhDFR gene expression than white flowers. (4) The prokaryotic expression vector pET-28a-RhDFR was successfully constructed and induced to express a protein with a size of about 38 kD, which was similar to the theoretical value. It is believed that the function of DFR enzyme activity in Rhododendron is diversified, and the DFR gene is related to the color of R. hybridum Hort. petals.