Since the discovery of single-layer graphene in 2004, the family of 2D inorganic nanosheets is considered as ideal membrane materials due to their ultrathin atomic thickness and fascinating physicochemical properties. However, the intrinsically nonporous feature of 2D inorganic nanosheets hinders their potential to achieve a higher flux to some extent. Recently, 2D polymer nanosheets, originated from the regular and periodic covalent connection of the building units in 2D plane, have emerged as promising candidates for preparing ultrafast and highly selective membranes owing to their inherently tunable and ordered pore structure, light weight, and high specific surface. In this review, the synthetic methodologies (including top-down and bottom-up methods) of 2D polymer nanosheets are first introduced, followed by the summary of 2D polymer nanosheets-based membrane fabrication as well as membrane applications in the fields of gas separation, water purification, organic solvent separation, and ion exchange/transport in fuel cells and lithium-sulfur batteries. Finally, based on their current achievements, the authors' personal insights are put forward into the existing challenges and future research directions of 2D polymer nanosheets for membrane separation. The authors believe this comprehensive review on 2D polymer nanosheets-based membrane separation will definitely inspire more studies in this field.