<p>Soluble amyloid-β oligomers (AβOs) are a hypothesized source of neurotoxicity in Alzheimer disease. Binding proteins that recognize these species may have high utility in diagnostic and therapeutic applications. However, identifying binders to AβOs directly generated from the aggregation cascade is challenging because of the short lifetime and low concentrations of oligomer populations. We report a strategy to detect binding to AβOs formed during Aβ42 aggregation using a genetically encoded biosensor. We show that our method enables rapid, highly reproducible measurement of the activity of existing AβO binders and can be used to select for new binders with improved potency. We uncover hits that are &gt;20-fold more effective than reported binders at delaying secondary nucleation, the step in Aβ aggregation thought to generate the highest number of toxic oligomers. Our approach may greatly accelerate the discovery and characterization of binding proteins that target AβOs.</p><p></p>