Isoquercitrin is a rare flavonol glycoside with a wide range of biological activities and is a key synthetic intermediate for the production of enzymatically modified isoquercitrin. In order to establish an ultrafast biopro-cess for obtaining isoquercitrin, a novel continuous flow biosynthesis of isoquercitrin using the hesperidinase-catalyzed hydrolysis of rutin in a glass–polydimethylsiloxane (PDMS) microreactor was first performed. Using the developed microchannel reactor (200 μm width, 50 μm depth and 2 m length) with one T-shaped inlet and one outlet, the maximum yield of isoquercitrin (98.6 %) was achieved in a short time (40 min) under the following optimum conditions: rutin concentration at 1 g L-1, hesperidinase concentration at 0.1 g mL-1, reaction temperature 40 °C, and a flow rate of 2 μL min-1. The value of the activation energy, Ea, of the enzymatic reaction was 4.61 kJ mol-1, and the reaction rate and volumetric productivity were approximately 16.1-fold and 30 % higher, respectively, than those in a batch reactor were. Thus, the use of a continuous-flow microreactor for the enzymatic hydrolysis of rutin is an efficient and simple approach to achieve a relatively high yield of isoquercitrin.