In recent years, the research on bitumen has reached the nanometer level, but there are still some problems in the study of the relationship between the atomic force microscope (AFM) nano-morphology and ageing of bitumen. The purpose of this paper is to find out the effect of thermal oxygen ageing on the nano-morphology of bitumen. Atomic force microscope (AFM) test sample of bitumen was prepared in this paper. Area ratio of bee-like structure, roughness, and maximum amplitude were selected as the nano-morphological parameters of the bitumen. The calculation method of nano-parameters was also proposed. Bitumen with different ageing degrees was prepared by thin film oven test (TFOT). Nano-morphological parameters of bitumen with different ageing degrees were obtained by AFM and analyzed with NanoScope Analysis, and the relationships between nano-morphological parameters and bitumen technical indexes after ageing, such as penetration, ductility, softening point, and viscosity were analyzed. The test results show that the wave crests and wave troughs of the bee-like structure on the surface of the bitumen alternate, while the other areas are relatively flat. The bee-like structure continues to develop as the ageing time prolongs, and the number of bee-like structures decreases, but the volume and the undulation degree increase. With the ageing time prolonging, the nano-parameters of bitumen, such as the area ratio of bee-like structure, roughness, and maximum amplitude, show a trend of increasing gradually, but the increasing rates become smaller and smaller. During the ageing process of bitumen, with the increase of nano-morphological parameters (area ratio of bee-like structure, roughness, and maximum amplitude), penetration at 25 °C and ductility at 15 °C of the bitumen decrease, and softening point and viscosity at 60 °C increase. AFM testing technology and image analysis method in this paper can be used for a reference in the nano-scale study of bitumen.