Sagittatoside B, a rare secondary flavonol glycoside in Epimedii Folium, has much better in vivo bioactivities than its original glycoside epimedin B. Its preparation methods, such as acidic hydrolysis, are of low efficiency, and byproducts are generated. The objective of this study was to establish a novel catalysis system for convenient preparation of this compound based on recyclable and integrated biphase enzymatic hydrolysis. beta-glucanase was selected from five commercial enzymes based on the best catalysis performance. After optimization of the conditions, the biphase system was constructed with propyl acetate and HAc-NaAc buffer (pH 4.5) (1:1, v/v) containing beta-glucanase/epimedin B (1:2, w/w), and the hydrolysis was performed at 60 degrees C for 1 h. Consequently, epimedin B was completely hydrolyzed to sagittatoside B, and 95.7% of the product was transferred into the organic phase. Moreover, a high conversion ratio of 94.0% was achieved, even after the enzyme solution was used for six cycles. Additionally, the procedure was much simplified compared with conventional enzymatic hydrolysis. The newly proposed strategy is an efficient and promising approach for the preparation of sagittatoside B in industrial applications.