An efficient isomerization catalyst is essential for the preparation of fructose from glucose, which is also an effective way to valorize cellulose to value-added chemicals. In this work, the interaction between catalytically active substances and biochar and the chemical properties of active sites are regulated by improving the oxygen content in biochar to enhance the efficiency of glucose isomerization catalyst. The biochar-based magnesium (Mg-BC) catalysts have been prepared by impregnation-pyrolysis complex with MgCl2 and biochar. Results indicate that the catalytic efficiency and active site stability of Mg-BC catalysts can be effectively improved by adjusting the oxygen content of biochar. The amounts of strongly basic active sites (48.69 mmol·g−1) in the Mg-BC-3 catalyst prepared by BC-3 with high oxygen content are obviously higher than that of the Mg-BC-1 catalyst (19.32 mmol·g−1) prepared by BC-1 with low oxygen content. And the large number of strongly basic active sites in Mg-BC-3 are more favorable for increasing glucose conversion and fructose selectivity, which can obtain efficiencies comparable to those of isomerase catalysts such as 48.15% glucose conversion, 85.38% fructose selectivity, and 41.11% fructose yield at 110 °C for 20 min. Meanwhile, a confinement effect of the mesoporous size of the catalyst on the active substance MgO and a relatively stable Mg-O-C group formed by the BC-3 and Mg2+ combine to improve the stability of the catalyst. Based on the density functional theory analysis, compared with the catalytic process without active sites, the loaded active substance MgO can effectively reduce the energy barrier from 67.1 to 21.3 kcal·mol-1 to form the enediol intermediates, further demonstrating the high catalytic efficiency of Mg-BC-3. In this work, we develop a highly efficient Mg-BC-3 catalyst for glucose isomerization and provide an efficient method for cellulose valorization.