High-pressure reactive distillation is a new bio-oil upgrading technology that can achieve high yields of the distillate due to reduced polymerization. Levoglucosan, as an anhydrous sugar, is one of the major components of bio-oil from the pyrolysis of biomass. To understand the reactive distillation of bio-oil at high pressure, the reactions and distribution of levoglucosan during the high-pressure reactive distillation of bio-oil were investigated in this study. Our results indicate that levoglucosan would mainly undergo hydrolysis during the high-pressure distillation, whereas thermal polymerization would dominate during the atmospheric pressure distillation. The reaction environment plays an important role in the levoglucosan reaction. The increase in pressure, temperature, water, and acetic acid concentration could accelerate levoglucosan conversion during bio-oil distillation, especially via the hydrolysis reaction. Most of the levoglucosan during the high-pressure reactive distillation could be converted into small molecules and distilled out due to the presence of light components retained in the liquid phase by high pressure. However, during the atmospheric pressure distillation, most of the levoglucosan would be mainly retained in the heavy residue to undergo polymerization reactions.