The amphipacity of the natively unstructured amyloid-β (Aβ40) peptide may play an important role in its aggregation into β-sheet rich fibrils that is associated with the pathogenesis of Alzheimer's disease. Using the air/water interface as an ideal hydrophobic interface, we characterized Aβ's surface activity and the structure, assembly, and morphology of Aβ adsorbed to the air/water interface. Aβ readily adsorbed to the air/water interface to form a contiguous film with a surface pressure of approximately 14 mN/m and showed an apparent critical micelle concentration of about 100 nM. Adsorbed Aβ was a single molecular layer extending ~ 20 Å into the aqueous subphase with in-plane ordering which gave rise to X-ray diffraction peaks. Analysis of the diffraction peaks showed that the air/water interface induced Aβ peptides to self-assemble into nano-size clusters with Aβ peptides folded in a β-sheet conformation. The presence of these clusters was further confirmed by imaging the morphology of the Aβ film using atomic force microscopy. The formation of these clusters were not affected by solution pH or ionic strength, or the presence of cosolutes sucrose and urea at concentrations known to stabilize and denature protein native structure in solution, suggesting that the hydrophobic interface-driven Aβ folding and assembly is robust and thereby strongly favorable. Furthermore, Aβadsorbed at the air/water interface can seed fibril growth in solution when re-introduced into the bulk. Our results implicate that that interface-induced Aβ folding and self-assembly may serve as a mechanism by which Aβ aggregation occurs in vivo.