Molecular chirality is introduced at liquid–solid interfaces. A ring-like aggregation of amyloid Aβ(1–40) on N-isobutyryl-L-cysteine (L-NIBC)-modified gold substrate occurs at low Aβ(1–40) concentration, while D-NIBC modification only results in rod-like aggregation. Utilizing atomic force microscope controlled tip-enhanced Raman scattering, we directly observe the secondary structure information for Aβ(1–40) assembly in situ at the nanoscale. D- or L-NIBC on the surface can guide parallel or nonparallel alignment of β-hairpins through a two-step process based on electrostatic-interaction-enhanced adsorption and subsequent stereoselective recognition. Possible electrostatic interaction sites (R5 and K16) and a chiral recognition site (H14) of Aβ(1–40) are proposed, which may provide insight into the understanding of this effect.