A series of Fe/beta zeolite catalysts with various Fe/Al ratios were prepared by liquid ion-exchanged method and were characterized by N2 adsorption, XRD, UV-Vis DRS, EPR, NH3-TPD, and FTIR of pyridine adsorption techniques. The catalysts were used to catalyze the transformation of glucose into 5-hydorxymethyfural ( HMF) in a biphasic reaction system under mild reaction conditions. The Fe/beta catalysts with Lewis acidic Fe sites is able to effectively improve the HMF yield compared to H-beta zeolite under identical reaction conditions, which is ascribed to the enhanced isomerization activity of glucose-to-fructose by Lewis acidic Fe species. Moreover, the Fe/Al ratio had a remarkable impact on the conversion of glucose and the yield of HMF, indicating that the synergetic effect of Lewis and Bronsted acid sites is critical to achieving high HMF yield. Under optimal reaction conditions, Fe/beta-0.06 catalyst afforded 61% HMF yield at glucose conversion of 95% after the reaction of 90 min at 120 degrees C, which is one of the most effective catalysts for the conversion of glucose into HMF in a biphasic medium up to now. Furthermore, extra-framework isolated Fe species bound to the framework Al sites, are most likely the active sites for the isomerization of glucose-to-fructose in the conversion of glucose. The catalysts were very stable and could be reused at least five runs without significant loss in the catalytic activity.