Peptide and protein fibrils have attracted an enormous amount of interests due to their relevance to many neurodegenerative diseases and their potential applications in nanotechnology. Although twisted fibrils are regarded as the key intermediate structures of thick fibrils or bundles of fibrils, the factors determining their twisting tendency and their handedness development from the molecular to the supramolecular level are still poorly understood. In this study, we have designed three pairs of enantiomeric short amphiphilic peptides: (I3K)-I-L-K-L and (I3K)-I-D-K-D, (I3K)-I-L-K-D and (I3K)-I-D-K-L, and (I3K)-I-La-K-L and I-Da(3) K-D, and investigated the chirality of their self-assembled nanofibrils through the combined use of atomic force microscopy (AFM), circular dichroism (CD) spectroscopy, scanning electron microscopy (SEM), and molecular dynamic (MD) simulations. The results indicated that the twisted handedness of the supramolecular nanofibrils was dictated by the chirality of the hydrophilic Lys head at the C-terminal, while their characteristic CD signals were determined by the chirality of hydrophobic Ile residues. MD simulations delineated the handedness development from molecular chirality to supramolecular handedness by showing that the beta-sheets formed by (I3K)-I-L-K-L, (I3K)-I-La-K-L, and (I3K)-I-D-K-L a exhibited a propensity to twist in a left-handed direction, while the ones of (I3K)-I-D-K-D, (I3K)-I-Da-K-D, and (I3K)-I-L-K-D in a right-handed twisting orientation.