In the presented work, a series of Cr/beta zeolites were prepared and used as bifunctional catalysts with Bronsted and Lewis acidity in the conversion of biomass-derived carbohydrates to 5-hydroxymethylfurfural (HMF). The physicochemical properties of all catalysts were characterized by ICP-AES, N-2 physisorption, solid-state Al-27 NMR, XRD, SEM-EDX, UV-Vis, XPS, Raman, NH3-TPD and Py-FTIR techniques. It was found that the catalysts with appropriate total acidity, ratio of Lewis to Bronsted acid sites and acid density appear to be beneficial for HMF formation. Under mild reaction conditions, a superior 72% HMF yield with. 83% selectivity was achieved from glucose in the H2O/THF biphasic system by using the 0.4-Cr/beta zeolite catalyst. Moreover, the results of the stability evaluation showed that the catalytic activity of the 0.4-Cr/beta zeolite was not significantly lost after four consecutive cycles, and the framework remained intact. Therefore, the catalytic approach proposed in this study is believed to provide potential prospects for the production of HMF from biomass material refining in the future.