Glycosylation is an effective way to promote the total intake of polyphenols in humans by increasing the solubility of polyphenols. In this study, an efficient glycosylation system was built via the dynamic complexation of CD with polyphenols and synchronous coupling reaction with cyclodextrin glycosyltransferase (CGTase) in water. The glycosylation efficiencies of quercetin, naringenin, rutin, resveratrol and caffeic acid were 20.9, 3.6, 2.7, 3.4 and 1.5 times higher than the non-complexed system. To quantify conversion rate and determine the rate-limiting step, the mixed product was treated with amyloglucosidase to obtain α-glucosyl rutin, which was identified as rutin 4”-O-α-D-glucopyranoside with purity of 93.6 % and yield of 34.8 % from NMR, MS and HPLC analysis. The results of half-reaction kinetics showed that the catalytic efficiencies of ring-opening of γ-CD (k1) and glycosylation reaction of rutin (k2) were 621.92 and 9.43 mM−1·s−1. The rate-limiting step was clarified for the first time, showing that the ring-opening ability of CGTase to CD was much higher than its glycosylation ability to polyphenols. It is speculated that the rapid ring-opening reaction of CD affected its dynamic complexation, releasing many polyphenols which were not utilized by CGTase in time. Therefore, adjusting the ratio and concentration of CD resulted in an optimal glycosylation molar yield of 84.1 % for rutin, which was the highest yield reported so far in water. This study established a universal system and clarified the rate-limiting step in the enzymatic glycosylation, providing theoretical guidance for efficient production of polyphenol glycosylation.