Display omitted ► An effective approach was developed to isolate solvent-stable glycosidase. ► The solvent-stable β-fructosidase with acceptor specificity of target substrate. ► Highly efficient synthesis of puerarin glycoside was achieved in nonaqueous media. ► The β-fructosidase did not hydrolyze almost formed product using sucrose as donor. An effective approach was successfully developed to isolate glycosidase with resistance of hydrophilic organic solvent, simultaneously with acceptor specificity of the target substrate. By this approach, an efficient solvent tolerant glycosidase producing bacterium Arthrobacter nicotianae XM6 was obtained. The β-fructosidase from strain XM6 shows high activity and stability in 10–25% DMSO and 10–20% methanol with 90–99% yields of puerarin glycosides. The addition of hydrophilic solvents not only greatly promoted the solubility of puerarin, but also regulated main products from multifructosyl puerarin to monofructosyl puerarin with increasing solvent concentration. Extraordinary highly efficient synthesis of puerarin glycosides (111.3g/L of monofructosyl puerarin and 35.6g/L of difructosyl puerarin) was attained in 25% DMSO solvent system from 110.4g/L puerarin, which resulted a great facility for purification in large-scale process. The most novelty was that the β-fructosidase did not hydrolyze almost the newly formed glycosides using simply sucrose as donor.