Background: 2″-O-rhamnosyl icariside II, a rare secondary flavonol glycoside in Epimedii Folium (EF), has much better in vivo bioactivities than its original glycoside epimedin C. Its preparation methods, such as acidic hydrolysis, are of low efficiency, with by-products generated. Objective: The objective of this study was to establish a novel catalysis system for convenient preparation of this compound based on the recyclable and integrated biphase enzymatic hydrolysis. Materials and Methods: β-dextranase was selected from five commercial enzymes due to the best catalysis performance. After optimization of conditions, the biphase system was constructed with propyl acetate and HAc-NaAc buffer (pH 4.5) (3:2, v/v) containing β-dextranase/epimedin C (1:2, w/w), and the hydrolysis was performed at 60°C for 40 min. Results: Epimedin C was completely hydrolyzed to 2″-O-rhamnosyl icariside II, and 93.38% of the product has been transferred into organic phase; moreover, a high conversion rate had been achieved at 91.69% even after the enzyme solution was used for four cycles. In addition, the procedure was much simplified compared with conventional enzymatic hydrolysis. Conclusion: The newly proposed strategy is an efficient and promising approach for the preparation of 2″-O-rhamnosyl icariside II in industrial application.