Ascorbic acid, which is widely used as a therapeutic agent for various disorders (e.g. chronic diseases and cancers), has potential therapeutic roles for neurodegenerative disease such as Huntington’s, Parkinson’s and Alzheimer’s diseases. The ability to impede amyloid fibril formation has a great demand on developing clinical medicine with respect to successfully preventing neurodegenerative diseases. Here, we report that L-ascorbic acid inhibits β-lactoglobulin amyloid formation in vitro. For quantitative characterization of the inhibitory effect of L-ascorbic acid on fibrillation, we performed high-resolution atomic force microscopy and thioflavin T fluorescence assay. Fourier transform infrared spectra indicated secondary structure differences of fibrils formed with and without ascorbic acid. These results suggest great potential of ascorbic acid for use in the prevention or treatment of amyloidogenic diseases.