An ambient water nanofilm condensed on a solid surface provides a good model system to study the self-assembling behaviors of peptides in a confined environment In this paper the self-assembly of three short amyloid-related peptides in a water nanofilm confined on a mica substrate was studied using drying microcontact printing (D-mu CP) and atomic force microscopy (AFM) The three peptides which share the same amino acid sequence but have different terminal groups were placed on mica surfaces by D-mu CP The samples were then incubated in a chamber with a controlled temperature and relative humidity (RH) in which water nanofilms were generated on the sample surfaces AFM images revealed that the peptides assembled into two kinds of supramolecular structures nanofilaments and nanosheets The peptides' terminal groups and the thickness of the water nanofilms determined the self-assembled supramolecular structures in the water nanofilm Through AFM investigation of the formation and transformation of the peptides' supramolecular structures, we conclude that the peptides self-assembly process was dominated by weak interactions, such as hydrophobic and electrostatic interactions and hydrogen bonding, between the peptide molecules the mica substrate, and the water nanofilm On the basis of these results, a model that describes the peptide arrangement in the confined water nanofilm is proposed This study reveals the complicated interactions of the peptides at an interface, which may be a general mechanism in vivo because water confinement around biomolecules and membranes is a universal phenomenon