Anthocyanin, a water-soluble flavonoid pigment, serves as a key secondary metabolite and plays a major role in the formation of color in plant flowers, fruits and vegetables. Dihydroflavonol 4-reductase (DFR) is a key enzyme in the anthocyanin biosynthesis pathway, catalyzing the reduction of dihydroflavonols into leucoanthocyanidins. In this study, we presented the identification of a putative IlDFR gene from Iris lactea Pall var. chinensis. The amino acid sequences of IlDFR shares an evolutionary lineage among its same genus, and IlDFR showed high activity when dihydromyricetin (DHM) was used as a substrate, while less or no activity using dihydrokaempferol (DHK) or dihydroquercetin (DHQ) as a substrate in the enzymatic assay. This may be the reason way the I. lactea exhibits blue-purple color because it mainly biosynthesizes and accumulates delphinidins in its petals. The IlDFR expressed in a white flower variety of Petunia×hybrida converted noticeable different phenotypes that exhibited light to dark purple in their flowers. In a transgenic plant with dark purple flower and the highest expression level of IlDFR showed that the contents of delphinidin-based anthocyanins, including delphinidin and its methylated derivative petunidin, as well as their glycosides (glucoside, rutinoside, galactoside and sophoroside) were significantly in higher levels than those of no-transgenic negative control. These results further strengthened the evidence that IlDFR prefers DHM substrate. Our research will provide new gene resources and a basis for color modification of flowers, fruits and vegetables using molecular biology and genetic engineering techniques.