This paper investigated the nanostructure and oxidation reactivity of soot generated from biofuel 2,5-dimethylfuran pyrolysis with different CO additions and different temperatures in a quartz tube flow reactor. The morphology and nanostructure of soot samples were characterized by a low and a high resolution transmission electron spectroscopy (TEM and HRTEM) and an X-ray diffraction (XRD). The oxidation reactivity of these samples was explored by a thermogravimetric analyzer (TGA). Different soot samples were collected in the tail of the tube. With the increase of temperature, the soot showed a smaller mean particle diameter, a longer fringe length, and a lower fringe tortuosity, as well as a higher degree of graphization. However, the variation of soot nanostructures resulting from different CO additions was not linear. Compared with 0%, 50%, and 100% CO additions at one fixed temperature, the soot collected from the 10% CO addition has the highest degree of graphization and crystallization. At three temperatures of 1173 K, 1223 K, and 1273 K, the mean values of fringe length distribution displayed a ranking of 10% CO >100% CO >50% CO while the mean particle diameters showed the same order. Furthermore, the oxidation reactivity of different soot samples decreased in the ranking of 50% CO addition>100% CO addition>10% CO addition, which was equal to the ranking of mean values of fringe tortuosity distribution. The result further confirmed the close relationship between soot nanostructure and oxidation reactivity.