Fructose, glucose, and mannose were treated with subcritical aqueous ethanol for ethanol concentrations ranging from 0 to 80% (v/v) at 180–200 °C. The aldose–ketose isomerization was more favorable than ketose–aldose isomerization and glucose–mannose epimerization. The isomerization of the monosaccharides was promoted by the addition of ethanol. In particular, mannose was isomerized most easily to fructose in subcritical aqueous ethanol. The apparent equilibrium constants for the isomerizations of mannose to fructose, Keq,M→F, and glucose to fructose, Keq,G→F, were independent of ethanol concentration and increased with increasing temperature. Moreover, the Keq,M→F value was much larger than the Keq,G→F value. The enthalpies for the isomerization of mannose to fructose, ΔHM→F, and glucose to fructose, ΔHG→F, were estimated to be 18 and 24 kJ/mol, respectively, according to van’t Hoff equation. Subcritical aqueous ethanol can be used to produce fructose from glucose and mannose efficiently.