Free-standing 2D nanoassemblies are ultrathin nanomembranes or nanosheets constructed from constituent nanoscale building blocks including metal nanoparticles, quantum dots, magnetic nanoparticles, typically by a bottom-up self-assembly approach. Such free-standing nanoassemblies are a new class of advanced functional materials that can integrate unique optoelectronic properties of nanomaterials with thin film mechanics into confined 2D space. This offers attributes such as minimizing substrate effects, facile transfer and soft devices in This article is protected by copyright. All rights reserved. 2 comparison to the corresponding substrate-supported system. This Review covers the recent progress in fabrication, characterization and application of the free-standing 2D nanoassemblies. To begin, the attributes of free-standing 2D nanoassemblies are discussed, followed by the description of fabrication methodologies. Then their novel optical, electronic, mechanical, magnetic, and stimuli responsible properties are covered, and their potential applications in filtration membrane, nanomechanical devices, and chemical sensing are further discussed. Finally, perspectives on the challenges and future opportunities of the freestanding 2D nanoassemblies are shared.