In the past three decades, researchers have engaged in the relationship between the composition, macro performance, and microstructure of asphalt. There are many research results in the use of atomic force microscopy (AFM) to study the microstructure and related mechanisms of asphalt. Based on previous studies, the performance of asphalt from its microstructure has been observed and analyzed, and different evaluation indices and modification methods have been proposed, providing guidance toward improving the performance of asphalt materials and benefiting potential applications. This review focuses on the typical application and analysis of AFM in the study of the aging regeneration and modification properties of asphalt. Additionally, this review introduces the history of the rheological and chemical testing of asphalt materials and the history of using AFM to investigate asphalt. Furthermore, this review introduces the basic principles of various modes of application of AFM in the microstructure of asphalt, providing a research direction for the further popularization and application of AFM in asphalt or other materials in the future. This review aims to provide a reference and direction for researchers to further popularize the application of AFM in asphalt and standardize the testing methods of AFM. This paper is also helpful in further exploring the relationship between the microstructure and macro performance of asphalt.