Atomic force microscopy (AFM), as a type of scanning probe microscopy (SPM), possesses formidable capabilities for nanoscale imaging and force spectroscopy. Due to its advantages such as high resolution, nondestructive detection, minimal environmental restrictions, strong versatility, and real-time in situ analysis, AFM has become an indispensable tool in surface science and materials research, finding extensive applications in the study of the membrane separation and fouling processes. The tremendous advantages of AFM in characterization applications stem from its diverse tip functionalization techniques. This review encompasses the preparation of AFM probe tips and the modification techniques of special tips, including carbon nanotube (CNT) probes, metal nanowire probes, colloidal probes, and single-cell/molecule probes. Furthermore, it highlights the applications and advancements of AFM and probe modification techniques in membrane technology research. With the continuous development of tip modification techniques, the analytical capabilities of AFM will be further expanded, promising broader prospects for its application in the study of membrane fouling mechanisms and the development of antifouling membrane materials.