Heavy fraction of bio-oil, the bottom-layer of biomass fast pyrolysis oil, is hard to be directly used due to its high molecular weight, complex components and deteriorate thermal stability. Here, a novel catalytic hydrotreating process for the heavy fraction of bio-oil, including depolymerization, decarboxylation, decarbonylation, hydrogenation and hydrodeoxygenation (HDO), is proposed with Ru/alpha-Al2O3 catalyst and super/subcritical ethanol. Results demonstrated that heavy fraction of bio-oil was depolymerized into monomeric phenolic compounds, and then was converted to hydrocarbons by hydrodeoxygenation in one-pot. The average molecular weight (M-w) of upgraded oil decreased drastically from 8941 g/mol to 937 g/mol during the catalytic hydrotreating process, and the formation of coke is suppressed. Under the optimal conditions, the total yield of hydrocarbons, which were comprised of alkyl-substituted cyclohexane and alkyl-substituted benzene, could be up to 23.15%. These hydrocarbons have a high octane number, would be the desirable components for fungible liquid transportation fuel. Furthermore, Ru/a-Al2O3 catalyst can be regenerated by calcination in the presence of O-2. These results indicated high quality fuels could be obtained from heavy fraction of bio-oil over suitable catalytic system.