Diphenylalanine (FF) nanotubes are a star material in the field of peptide self-assembly and have demonstrated numerous intriguing applications. Due to its resemblance to amyloid assembly, the FF nanotube is widely regarded as a simplified mimic of amyloids. Yet, whether FF nanotube truly possesses amyloid structure remains an open question. To better understand the structural nature of FF nanotube, we herein performed a comparative structural investigation between FF nanotube and typical amyloid systems by Aβ1-40, Aβ1-42, Aβ16-22, Aβ13-23, α-synuclein, and lysozyme using Fourier transform infrared spectroscopy. Through this comparative investigation, we obtained clear evidence to support that the FF nanotube does not possess a β-sheet structure, a key structural characteristic of amyloid assembly, thus revealing the non-amyloid structural nature of the FF nanotube. At last, in light of our new finding, we further discussed the unique self-assembly behaviors of FF during nanotube formation and the implications of our work for FF nanotube related applications.