β-amyloid (Aβ) has been suggested as a potent neurotoxic agent. The Aβ-targeted immunotherapy aims to clear diffuse amyloid deposits and reverse memory deficits in Alzheimer's disease. We generated a human single chain variable domain antibody fragment (scFv) against Aβ40, termed E3, by screening a phage antibody library. E3 scFv could recognize Aβ in human cerebral cortex. It was able not only to prevent the aggregation of Aβ but also to disrupt the Aβ preexisting fibrils. Moreover, the Aβ toxicity to SK-N-SH cells was attenuated by addition of E3 scFv. Our results indicate that site-directed human scFv might be a potential therapeutic agent for Alzheimer's disease.