The variation of amyloid β peptide (Aβ) concentration and Aβ aggregation are closely associated with the etiology of Alzheimer's diseases (AD). The interaction of Aβ with the monosialoganglioside-rich neuronal cell membrane has been suggested to influence Aβ aggregation. Therefore, studies on the mechanism of Aβ and sialic acids (SA) interaction would greatly contribute to better understanding the pathogenesis of AD. Herein, we report a novel approach for Aβ-SA interaction analysis and highly sensitive Aβ detection by mimicing the cell surface presentation of SA clusters through engineering of SA-modified peptide nanofiber (SANF). The SANF displayed well-ordered 1D nanostructure with high density of SA on surface. Using FAM-labeled Aβ fragments of Aβ1-16, Aβ16-23, and Aβ24-40, the interaction between Aβ and SA was evaluated by the fluorescence titration experiments. It was found that the order of the SA-binding affinity was Aβ1-16 > Aβ24-40 > Aβ16-23. Importantly, the presence of full-length Aβ1-40 monomer triggered a significant fluorescence enhancement due to the multivalent binding of Aβ1-40 to the nanofiber. This fluorescent turn-on response showed high selectivity and sensitivity for Aβ1-40 detection and the method was further used for Aβ aggregation process monitoring and inhibitor screening. The results suggest the proposed strategy is promising to serve as a tool for mechanism study and the early diagnosis of Alzheimer's disease.