Free-standing 2D nanoassemblies are ultrathin nanomembranes or nanosheets constructed from constituent nanoscale building blocks including metal nanoparticles, quantum dots, and 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 the 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 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 with, 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 free-standing 2D nanoassemblies are shared.