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Isorhamnetin-3-<i>O</i>-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from <i>Arabidopsis thaliana</i> was cloned, expressed, and characterized in <i>Escherichia coli</i>. The optimal activity was at pH 7.0 and 45 &#176;C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 &#176;C. The <i>V_max</i> and <i>K_m</i> for isorhamnetin were 0.646 U/mg and 181 &#956;M, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 &#176;C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3-<i>O</i>-rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3-<i>O</i>-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-<i>O</i>-rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids.