Thin-film composite (TFC) polyamide (PA) membrane has been widely applied in nanofiltration, reverse osmosis, and forward osmosis, including a PA rejection layer by interfacial polymerization on a porous support layer. However, the separation performance of TFC membrane is constrained by the trade-off relationship between permeability and selectivity. Although thin-film nanocomposite (TFN) membrane can enhance the permeability, due to the existence of functionalized nanoparticles in the PA rejection layer, the introduction of nanoparticles leads to the problems of the poor interface compatibility and the nanoparticles agglomeration. These issues often lead to the defect of PA rejection layers and reduction in selectivity. In this review, we summarize a new class of structures of TFN membranes with functionalized interlayers (TFNi), which promises to overcome the problems associated with TFN membranes. Recently, functionalized two-dimensional (2D) nanomaterials have received more attention in the assembly materials of membranes. The reported TFNi membranes with 2D interlayers exhibit the remarkable enhancement on the permeability, due to the shorter transport path by the “gutter mechanism” of 2D interlayers. Meanwhile, the functionalized 2D interlayers can affect the diffusion of two-phase monomers during the interfacial polymerization, resulting in the defect-free and highly crosslinked PA rejection layer. Thus, the 2D interlayers enabled TFNi membranes to potentially overcome the longstanding trade-off between membrane permeability and selectivity. This paper provides a critical review on the emerging 2D nanomaterials as the functionalized interlayers of TFNi membranes. The characteristics, function, modification, and advantages of these 2D interlayers are summarized. Several perspectives are provided in terms of the critical challenges for 2D interlayers, managing the trade-off between permeability, selectivity, and cost. The future research directions of TFNi membranes with 2D interlayers are proposed.