Janus particles are micro-nano materials with anisotropic surface chemical properties, exhibiting unique advantages in emulsion stabilization, interfacial adsorption, and selective wetting. The high interfacial activity of Janus particles makes them widely used in oil-water separation research and shows broad application prospects in environmental protection and other fields. In this perspective, we summarize the research progress of emerging Janus particles in preparation, properties, and oil-water separation applications in recent years. We focus on discussing the influence of different preparation strategies and morphologies of Janus particles on their interfacial properties, summarize the mechanism of particle assembly and aggregation at interface, and introduce various Janus particles with reversible responsive properties. Finally, we prospect the challenges and future development directions of Janus particles in oil-water separation, such as green synthesis, large-scale preparation, in-depth mechanism research, and applications in aspects such as micro-oil droplet removal, crude oil treatment, and controllable responsive separation.