A highly efficient and promiscuous 7,4′-di-O-glycosyltransferase ZjOGT3 was discovered from the medicinal plant Ziziphus jujuba var. spinosa. ZjOGT3 could sequentially catalyse 4′- and 7-O-glycosylation of flavones to produce 7,4′-di-O-glycosides with obvious regio-selectivity. For 7,4′-dihydroxyl flavanones and 3-O-glycosylated 7,4′-dihydroxyl flavones, ZjOGT3 selectively catalyses 7-O-glycosylation. The crystal structure of ZjOGT3 was solved. Structural analysis, DFT calculations, MD simulations, and site-directed mutagenesis reveal that the regio-selectivity is mainly controlled by the enzyme microenvironment for 7,4′-dihydroxyl flavones and 3-O-glycosylated 7,4′-dihydroxyl flavones. For 7,4′-dihydroxyl flavanones, the selectivity is mainly controlled by intrinsic reactivity. ZjOGT3 is the first plant flavonoid 7,4′-di-O-glycosyltransferase with a crystal structure. This work could help understand the catalytic mechanisms of multi-site glycosyltransferases and provides an efficient approach to synthesise O-glycosides with medicinal potential.