The aggregation of β-amyloid (Aβ) peptides to form amyloid plaques is one of the primary hallmarks for Alzheimer's disease (AD). Dietary flavonoid supplements containing hesperetin have an ability to decline the risk of developing AD, but the molecular mechanism is still unclear. In this work, hesperetin, a flavanone abundant in citrus fruits, has been proven to prevent the formation of Aβ aggregates and depolymerized preformed fibrils in a concentration-dependent fashion. Hesperetin inhibited the conformational conversion from the natural structure to a β-sheet-rich conformation. It was found that hesperetin significantly reduced the cytotoxicity and relieved oxidative stress eventuated by Aβ aggregates in a concentration-dependent manner. Additionally, the beneficial effects of hesperetin were confirmed in Caenorhabditis elegans, including the inhibition of the formation and deposition of Aβ aggregates and extension of their lifespan. Finally, the results of molecular dynamics simulations showed that hesperetin directly interacted with an Aβ42 pentamer mainly through strong non-polar and electrostatic interactions, which destroyed the structural stability of the preformed pentamer. To summarize, hesperetin exhibits great potential as a prospective dietary supplement for preventing and improving AD.