Robust polydiacetylene-based colorimetric sensing material has been developed with amyloid fibrils of α-synuclein in the presence of 10,12-pentacosadiynoic acid (PCDA) by taking advantage of the specific fatty acid interaction of α-synuclein and structural regularity of the self-assembled product of amyloid fibrils. PCDA facilitated not only self-oligomerization of α-synuclein but also its fibrillation into the fibrils with increased thickness. Upon UV irradiation, the PCDA-containing amyloid fibrils (AF-PCDAs) turned blue, which then became red following heat treatment. The blue-to-red color transition was also observed with other stimuli of pH and ethanol. AF-PCDAs were demonstrated to be mechanically stable since not only the individual colors of blue and red but also their colorimetric transition were not affected by a number of sonications which readily disrupted the polydiaceylene (PDA) vesicles with the instant loss of color. Therefore, AF-PCDA can be considered to be a novel PDA-based colorimetric sensing material with high mechanical strength, which has the potential to be employed in various areas involving advanced sensing technologies.