Amyloid fibrils are unique fibrous polypeptide aggregates. They have been associated with more than 20 serious human diseases including Alzheimer's disease and Parkinson's disease. Besides their pathological significance, amyloid fibrils are also gaining increasing attention as emerging nanomaterials with novel functions. Structural characterization of amyloid fibril is no doubt fundamentally important for the development of therapeutics for amyloid-related diseases and for the rational design of amyloid-based materials. In this study, we explored to use side-chain-based infrared (IR) probe to gain detailed structural insights into the amyloid fibril by a 21-residue model amyloidogenic peptide, A beta(8-28). We first proposed an approach to incorporate thiocyanate (SCN) IR probe in a site-specific manner into amyloidogenic peptide using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate as cyanylating agent. Using this approach, we obtained three A beta(8-28) variants, labeled with SCN probe at three different positions. We then showed with thioflavin T fluorescence assay, Congo red assay, and atomic force microscopy that the three labeled A beta(8-28) peptides can quickly form amyloid fibrils under high concentration and high salt conditions. Finally, we performed a detailed IR spectral analysis of the A beta(8-28) fibril in both amide I and probe regions and proposed a millipede-like structure for the A beta(8-28) fibril.