A new approach for studying a peptide conformation of amyloid fibril has been developed. It is based on infrared linear dichroism analysis using an IR-microscope for aligned amyloid fibril. The polarization directions of amide I and II bands were perpendicular similarly for β2-microglobulin and its #21?31 peptide. Furthermore, this approach has shown that the #21?31 peptide consists of two C=O bonds in the β-sheet that makes 0° with the fibril axis, three C=O bonds in the β-sheet inclined by 27° with respect to the fibril axis, four residues in the random coil by 47°, and two residues in possible β-bulge structure by 32°. Plausible structures of the amyloid core in the fibril are proposed by taking account of these results.