Isoorientin and isovitexin, kinds of flavone C-glycosides, exhibit a number of biological properties. In this work, The C-glucosyltransferase (Gt6CGT) gene from Gentiana triflora was cloned and expressed in Escherichia coli BL21(DE3). The optimal activity of Gt6CGT was at pH 7.5 and 50 degrees C. The enzyme was stable over pH range of 6.5-9.0, and had a 1-h half-life at 50 degrees C. The V-max for luteolin and apigenin was 21.1 nmol/min/mg and 31.7 nmol/min/mg, while the K-m was 0.21 mM and 0.22 mM, respectively. Then, we developed an environmentally safe and efficient method for isoorientin and isovitexin production using the coupled catalysis of Gt6CGT and Glycine max sucrose synthase (GmSUS). By optimizing coupled reaction conditions, the titer of isoorientin and isovitexin reached 3820 mg/L with a corresponding molar conversion of 94.7% and 3772 mg/L with a corresponding molar conversion of 97.1%, respectively. The maximum number of UDP-glucose regeneration cycles (RCmax) reached 28.4 for isoorientin and 29.1 for isovitexin. The coupled catalysis reported herein represents a promising method to meet industrial requirements for large-scale isoorientin and isovitexin production in the future.