Fructose is a vital ketose in the production of 5-hydroxymethylfurfural platform molecules. Herein, Zr-doped hexagonal mesoporous silica was prepared over in situ co-assembly (Zr-x-HMS, x is the mole ratio of Si to Zr, x = 20, 50) and post-impregnation (Zr-Imx-HMS, x = 20, 50, 100) processes to regulate its catalytic performance against the isomerization of glucose to fructose in ethanol. The post-impregnation process did not significantly change the original pore structure and morphology of HMS, Zr-Im50-HMS with medium Zr loading possessed the most Lewis acid sites with medium strength among Zr-Imx-HMS catalysts. The in situ process changed the physical properties of HMS and enhanced the interaction of Zr sites and silica framework, facilitating the formation of Lewis acid sites with medium strength. Zr-Im50-HMS possessed the most suitable acidity for fructose production, and an optimal yield of 31.2% was achieved at 160 °C and 20 min. Moreover, the recyclability of Zr-Im50-HMS was acceptable.