Membrane fouling presents a significant challenge in the treatment of wastewater. Several detection methods have been used to interpret membrane fouling processes. Compared with other analysis and detection methods, atomic force microscopy (AFM) is widely used because of its advantages in liquid-phase in situ 3D imaging, ability to measure interactive forces, and mild testing conditions. Although AFM has been widely used in the study of membrane fouling, the current literature has not fully explored its potential. This review aims to uncover and provide a new perspective on the application of AFM technology in future studies on membrane fouling. Initially, a rigorous review was conducted on the morphology, roughness, and interaction forces of AFM in situ characterization of membranes and foulants. Then, the application of AFM in the process of changing membrane fouling factors was reviewed based on its in situ measurement capability, and it was found that changes in ionic conditions, pH, voltage, and even time can cause changes in membrane fouling morphology and forces. Existing membrane fouling models are then discussed, and the role of AFM in predicting and testing these models is presented. Finally, the potential of the improved AFM techniques to be applied in the field of membrane fouling has been underestimated. In this paper, we have fully elucidated the potentials of the improved AFM techniques to be applied in the process of membrane fouling, and we have presented the current challenges and the directions for the future development in an attempt to provide new insights into this field.