The fractioned preparation of bio-oil by biomass pyrolysis in a vacuum pyrolysis apparatus and its characteristics were investigated. The off-gas was composed of CO2 and CO with 69.77 vol% and 30.23 vol% separately at the temperature of 200 degrees C. There were more volatile small molecules and high proportion of organic acids in the bio-oil produced below 300 degrees C, and much water alleviated the acidity. The maximum amount of bio-oils of total liquid product was obtained between 300 degrees C and 400 degrees C with 47.81 wt%, in which the big molecules, such as aromatic compounds, increased and volatile compounds decreased for the enhanced secondary cracking, so H2, CO, CH4, C2, and C3 rose remarkably. Above 500 degrees C, the phenols with many side chains were easier to crack, which makes phenol and 1,2-benzenediol augmented by degradation of 2-methoxy-phenol. The longer retention time could increase the bio-oil yield at 300 degrees C-500 degrees C. Biomass vacuum pyrolysis can centralize the similar components of bio-oil and realized the preliminary fractioned preparation.