This review comprehensively explores the remarkable progress in the application of atomic force microscopy (AFM) in extracellular vesicle (EV) research. AFM, with its unique capacity for high-resolution imaging and sensitive force measurement, has arisen as a potent method for studying the nanoscale environment. EVs, a mixed group of membrane vesicles produced by cells, are crucial in facilitating intercellular communication and disease progression. Here, we elucidate the fundamental principles of AFM and the characteristics of EVs. We then delve into the significant findings enabled by the combination of AFM with EV research, including the visualization of EV morphology, the measurement of mechanical properties, and the investigation of interactions with other molecules and cells. Additionally, we discuss the challenges and future directions in this burgeoning field. This review not only highlights the current state-of-the-art but also provides insights into the potential of AFM to further our understanding of EVs and their implications in biology and medicine.