Natural low-calorie rebaudioside M (Reb M), one of the most ideal sweeteners, was traditionally extracted from plants, which is time- and energy-consuming. The bioconversion of low-value rebaudioside A (Reb A) is green and economical. A series of hybrid enzymes of UGT76G1 and EUGT11 were designed and constructed to highly efficiently convert Reb A to Reb M. The effects of the order of EUGT11 and UGT76G1, as well as the length and amino acid composition of the linker on activity, were investigated, and UL4E containing N-terminal UGT76G1, an FRFRF linker, and C-terminal EUGT11 showed the best catalytic performance. The specific activity of the purified UL4E was 2.01×103 U/g. A low-cost system for Reb M synthesis was designed using BL21 (pET-UL4E) strain cells as biocatalysts. The cells were treated with various surfactants or freezing to improve permeability, with 0.3 g/L CTAB treatment proving optimal. A single-factor and orthogonal L9 (34) experiment was conducted, yielding a Reb M mass yield of 102.9% using 165 OD600 cells incubated at 34 °C for 10 hours at 100 rpm. Cell treatment conditions were further optimized using response surface methodology. When cells were suspended to 26 OD600 in 4 °C-precooled CTAB, incubated for 5 min, and immediately collected for use, Reb A was completely consumed, and the Reb M mass yield reached 109.4%. Therefore, our study successfully developed a novel cascade biocatalysis strategy and established an efficient biosynthesis system for glycosides production, thus providing a green and promising route for manufacturing of high-value glycosylated compounds.