Abstract Polypeptides and proteins have a high density of chemical functionality, which can be used to construct defined macromolecular nano architectures, such as low dimensional assemblies (i.e., nanoparticles, nanofibers and nanotubes), two-dimensional nanosheets, and extended three-dimensional structures (i.e., crystalline solids). Two-dimensional nanosheets are a fundamentally important geometry that bridges the gap between low dimensional assemblies and three-dimensional structures. In view of the considerable impact on a great many fundamental and applied aspects of biological and material sciences, it is therefore significant and valuable to review the recent works related to this field. In recent years, a central goal is to design novel materials with molecular-level information that can be utilized to direct highly specific intra- and intermolecular interactions promoting self-assembly of thermodynamically stable and structurally defined two-dimensional (2D) assemblies. In this review, we present a variety of strategies for constructing the 2D nanoscale assemblies and nanomaterials using specific interactions of polypeptides or similar peptoid molecules in a well-predictable manner. These controllable 2D nanomaterials can also display a variety of functionalities. Initially, the primary and secondary structure of peptides will be introduced. Examples of controlled fabrication of two-dimensional assemblies on the nanoscale are subsequently presented based on different secondary structures of the peptides, e.g. two-dimensional crystals from α-helix, nanoscale sheets from self-assembly of collagen-mimetic peptides with triple helix, co-assembled nanosheet structures assembled from peptide-organic molecules with β-strand conformation, and 2D crystals assembled from peptoid with Σ-strand structure. Furthermore, macrofilms assembled from protein fibril arrays are introduced. These bottom-up strategies can arrange polypeptides and proteins into well-ordered 2D structures, which can have wide-ranging applications, such as membrane-based separations with specific mechanical properties, the control of surface properties in nanodevice and nanosensor fabrication, retroviral transduction in biology and diagnosis of some disease in biomedicine.