The interaction of amyloid beta-peptide (A beta) with Cu2+ is crucial to the development of neurotoxicity in Alzheimer's disease (AD). Many recent studies show a variation on the dissociation constant of A beta-Cu2+ under different solvent conditions. Among various buffers, the Tris(hydroxymethyl)aminomethane (Tris) buffer is the most reliable chelator of Cu2+. However, as a typical nucleophilic reagent capable of binding peptides, the behavior of Tris should be more complicated. In this work, the effect of Tris on the interaction of A beta with Cu2+ was investigated. Under acidic conditions, Tris-A beta-Cu2+ ternary complex was identified by electrospray ionization mass spectrometry and transmission electron microscopy. The results of surface plasmon resonance reveal that the formation of the ternary complex increases the dissociation constant by almost 1 order of magnitude. Consequently, the assessment of toxicity indicates that the generation of center dot OH induced by the A beta-Cu2+ complex was enhanced in the presence of Tris. The work reveals the significant side effect of Tris on the interaction of A beta with Cu2+, which will greatly improve the quantitative investigation on A beta-Cu2+ interaction and be helpful for the in-depth understanding of the roles of A beta and Cu2+ in AD neuropathology.