The cause of the formation of amyloid-ß plaques, which play a critical role in neuronal death in Alzheimer's disease (AD), is unknown. The objective of this study was to investigate the level of protein unfolding in the sera of patients with AD. Sera were collected from young healthy adults, healthy older adults without evidence of cognitive impairment, patients with mild cognitive impairment (MCI), and patients with AD. We used a previously developed fluorescent liposome assay (Translational Medicine 2015; 5:1-9) to determine the albumin-fatty acid binding activity (Alb-FA-BA) as a marker of the level of protein unfolding; protein unfolding is recognized as an initial step involved in amyloid aggregation and plaque formation. The Alb-FA-BA was markedly augmented after albumin was exposed to increased temperature up to 80oC or to ß-mercaptoethanol. The increased Alb-FA-BA was likely due to increasing unfolded albumin forms that appeared under these conditions. Sera of patients with AD had significantly higher Alb-FA-BA than the healthy older control group (149.740 ± 3.805 vs. 130.821 ± 4.894, p = 0.0018, n = 24), the young healthy control group (123.051 ± 4.660, p < 0.0001, n = 25), and subjects with MCI (128.399 ± 3.520, p = 0.0005, n = 24). However, total serum albumin levels from all samples were approximately the same. A molecular weight (MW) < 10k factor in the serum was isolated to a partially pure form that stimulated Alb-FA-BA. The MW < 10k factor isolated from patients with AD stimulated Alb-FA-BA significantly more than the factor from the older control and MCI subject groups (161.146 ± 7.236, 124.385 ± 6.075, and 141.410 ± 4.897, respectively, p = 0.0124 for AD vs. older control, p = 0.0295 for AD vs. MCI) (n = 11 for each group). High Alb-FA-BA in the AD sera may indicate the presence of an increased content of unfolded albumin induced by the MW < 10k factor. The Alb-FA-BA and the MW < 10k factor could be useful biomarkers for AD.