Glucose isomerization to fructose is crucial for its application in food industry and biorefinery. Herein, strategies for developing MgO-biochar catalysts to convert glucose to fructose were explored and optimized with an emphasis on the influence of synthesis method on the stability of the catalyst. Despite the favorable activity exhibited by all catalysts, the embedding of MgO on the surface, loading onto porous structures, or incorporating into a carbon framework significantly diminished the catalytic activity and stability of magnesium species. The one-step synthesis approach facilitates the uniform distribution of MgO nanoparticles on the biochar matrix, enhancing their interactions and preventing Mg leaching compared to MgO-biochar prepared through two-step methods. After optimization, fructose yield exceeding 26% and a selectivity of 91% were obtained under mild conditions of 80 celcius for 2 h in water. In summary, the effective anchoring of MgO on the biochar exhibited dual effects for enhanced catalytic activity and stability.