Islet amyloid polypeptide (amylin) secreted from the pancreas crosses from the blood to the brain parenchyma and forms cerebral mixed amylin-β amyloid (Aβ) plaques in persons with Alzheimer’s disease (AD). Cerebral amylin-Aβ plaques are found in both sporadic and early-onset familial AD; however, the role of amylin-Aβ co-aggregation in potential mechanisms underlying this association remains unknown, in part due to lack of assays for detection of these complexes. Here, we report the development of an ELISA to detect amylin-Aβ hetero-oligomers in brain tissue and blood. The amylin-Aβ ELISA relies on a monoclonal anti-Aβ mid-domain antibody (detection) and a polyclonal anti-amylin antibody (capture) designed to recognize an epitope that is distinct from the high affinity amylin-Aβ binding sites. The utility of this assay is supported by the analysis of molecular amylin-Aβ codeposition in postmortem brain tissue obtained from persons with and without AD pathology. By using transgenic AD-model rats, we show that this new assay can detect circulating amylin-Aβ hetero-oligomers in the blood and is sensitive to their dissociation to monomers. This is important because therapeutic strategies to block amylin-Aβ co-aggregation could reduce or delay the development and progression of AD.