To develop a practical method to prepare tilianin by highly selective and efficient hydrolysis of the C-7 rhamnosyl group from linarin. Naringinase was utilized to selectively catalyze the formation of tilianin using linarin as the starting material. The reaction conditions, including temperature, pH, metal ions, substrate concentration and enzyme concentration, were optimized. At 60 A degrees C, naringinase showed enhanced alpha-l-rhamnosidase activity while the beta-d-glucosidase activity was abrogated. The addition of Mg2+, Fe2+ and Co2+ was also beneficial for selective biotransformation of linarin to tilianin. Under the optimized conditions (pH 7.0 at 60 A degrees C), linarin could be nearly completely transformed to tilianin with excellent selectivity (> 98.9 %), while that of the by-product acacetin was less than 1.1 %. In addition, the structure of target product tilianin was fully characterized by HR-MS and H-1-NMR. A highly selective and efficient biotransformation of linarin to tilianin was developed by the proper control of incubation temperature, which enhanced the alpha-l-rhamnosidase activity of naringinase and blocked its beta-d-glucosidase activity.