Abstract: As a star material, graphene has attracted much attention because of its excellent mechanical, optical, electrical, and thermal properties. The chemical conversion of graphene oxide is considered to be a promising approach to economically produce graphene in significant quantities, but the reduced graphene oxide prepared from this method suffers a lot of defects, such as vacancies and the presence of residual oxygen groups. In this case, graphite oxide with a low oxidation degree was used to prepare graphene to decrease the residual oxygen groups and vacancies caused by the removal of oxygen groups during reduction. However, this kind of mildly oxidized graphite (MOG) is hard to exfoliate to prepare graphene oxide; it is necessary to increase the basal spacing of MOG to facilitate the exfoliation. According to the literature, the intercalation of surfactants is usually used to enlarge the basal spacing of graphite and graphite oxide, but the intercalation mechanism of surfactants with different structures in MOG remains unknown. In this work, MOG was prepared from artificial graphite powder, and the intercalations of surfactants with different polar parts and carbon chain lengths on MOG were studied. The effect of surfactant molecular structures on their intercalation ability and the intercalation mechanism were studied through measurements with X-ray diffraction spectroscopy (XRD), Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and Raman and Zeta potential measurements. The results show that the cationic surfactants intercalate MOG mainly through the attractive electrostatic interaction between them, and their intercalation capacity is better than that of anionic surfactants as they can enlarge the basal spacing of MOG more easily. The anionic surfactants intercalated MOG through the formation of hydrogen bonds and the hydrophobic intercalation force between them. It was found that the larger the polar part and the longer the carbon chain, the stronger the intercalation ability of surfactants. These research results may help to better understand the intercalation mechanism of the surface in MOG interlayers and guide the preparation and application of MOG intercalation-modified materials.