Abstract: Bio-oil is one type of liquid product from the pyrolysis liquefaction of biomass. The heat-sensitive liquid with high water content and complex components can be further processed and then refined for the subsequent application. Moreover, the more large molecules of phenolics and sugars in the bio-oil are prone to serious coking on the catalyst during refinement. In this study, a systematic investigation was carried out on the distribution pattern of molecular distillation fractions in the different bio-oils. Two feedstocks (pine and maize stover) and temperatures (500, 550, and 600 ℃) were selected to evaluate the separation of bio-oils using molecular distillation. The pyrolysis liquefaction test was carried out by a bubbling fluidized bed reactor. The experiment showed that the bio-oil components were dominated by acids, aldehydes, ketones, phenols, and sugars. Both feedstock and pyrolysis temperature dominated the yield of bio-oil. Specifically, the yield of light components in pine bio-oil decreased continuously from 21% to 11% with the increase in pyrolysis temperature. While the yield of light components in maize stover bio-oil was stabilized at about 20%. There was a strong polarization in the mean free path of each component in the bio-oil at high temperatures. Once the pyrolysis temperature increased from 500 to 600 ℃, the evaporation percentage of pine light oil increased to 92%, while that of maize stover light oil increased to 86%. The molecular distillation was realized to separate more than 90% of water, acids, ketones and small molecules in the bio-oils. But it was difficult to separate the larger molecules of sugars. The separation of phenolics differed greatly from the number of phenolic hydroxyl groups; Monophenolics were easily vaporized, and thus more enriched in the distillation fraction (DF); While diphenolics were more difficult to be vaporized and thus more enriched in the residual fraction (RF). Higher pyrolysis temperatures promoted the conversion of methoxy into the phenolic hydroxyl groups in the bio-oil phenolics. The increasing number of phenolic hydroxyl groups in phenolics also resulted in the transfer of phenolics from the DF to the RF. The separation factor was introduced to evaluate the separation effect of various compounds in the bio-oil after molecular distillation. Among them, the separation factors of acids, ketones and mono phenolics in the bio-oil reached more than 0.9, whereas, less than 0.1 was found in the diphenolics of sugar. Therefore, the different compounds were enriched by more than 90% in their fractions. Molecular distillation was also used to increase the calorific value of bio-oils, where the water content and pH of bio-oils were reduced to obtain the higher-quality RF fractions. The effective separation was realized to improve the different bio-oils. The DF contained mainly acids, ketones and small-molecule phenols, while the RF was dominated by sugars and large-molecule phenols. The enrichment degree of most compounds in the bio-oils was more than 90%, except for the compounds, such as glycolaldehyde and benzofuran. This finding can provide a strong reference for the separation and subsequent refinement of bio-oil after biomass-fast pyrolysis.