A solvent-free ball milling protocol was investigated for synthesizing sustainable Sn-functionalized biochars for glucose isomerization to fructose. Raw wood biomass (W) and its derived biochars pyrolyzed at low (LB, 400 °C) and high (HB, 750 °C) temperatures were investigated as catalyst supports. This study emphasized that the interactions between Sn and the carbonaceous supports were related to the surface chemistry of the catalysts. Functional group-enriched surfaces provide more active sites for anchoring Sn, resulting in a high loading on the biochar support. Sn was primarily bound with W via surface complexation or precipitation, while it mainly interacted with LB and HB via physical adsorption. The annealing temperature was another critical factor that affected the concentrations and nature of the species of loaded Sn. Catalytic conversion experiments indicated that SnW annealed at 750 °C exhibited the best fructose yield (12.8 mol%) and selectivity (20.2 mol%) at 160 °C in 20 min. The catalytic activity was correlated to the amount and nature of active Sn sites. Reusability tests revealed a noticeable increase in product selectivity compared to pristine materials, despite a compromise in product yield. This study elucidated the roles of the carbon support and annealing temperature for synthesizing biochar-supported catalysts, highlighting a simple and green approach for designing effective solid catalysts for sustainable biorefineries.