Peptide amphiphile (PA) is a small molecule containing both hydrophilic and hydrophobic motifs in the peptide sequence. As it is designed from natural amino acids, it exhibits good biocompatibility, high bioactivity, and high biodegradability. Therefore, PA can be used as a flexible building block for the molecular design and utilization in various ways and has been widely used in various scientific and technical fields. In addition, PA shows the ability to self-assemble into various amyloid aggregates and can self-assemble into a series of functional nanostructures, such as nanofibers, nanotubes, spherical micelles, vesicles, nanosheets, and hydrogels. In this chapter, we focus on the design, synthesis, and self-assembly mechanisms of PAs. The effects of environmental adjusting, such as the interactions, pH, ionic strength, and temperature, on the self-assembly of PAs and the formation of supramolecular nanostructures are presented and discussed. This chapter will provide valuable information and inspirations for the rational design and synthesis of PA-based functional nanomaterials for biomedical and nanotechnological applications.