BackgroundThe current diagnosis of Alzheimer’s disease (AD) is based on a series of analyses which involve clinical, instrumental and laboratory findings. However, signs, symptoms and biomarker alterations observed in AD might overlap with other dementias, resulting in misdiagnosis.MethodsHere we describe a new diagnostic approach for AD which takes advantage of the boosted sensitivity in biomolecular detection, as allowed by seed amplification assay (SAA), combined with the unique specificity in biomolecular recognition, as provided by surface-enhanced Raman spectroscopy (SERS).ResultsThe SAA-SERS approach supported by machine learning data analysis allowed efficient identification of pathological Aβ oligomers in the cerebrospinal fluid of patients with a clinical diagnosis of AD or mild cognitive impairment due to AD.ConclusionsSuch analytical approach can be used to recognize disease features, thus allowing early stratification and selection of patients, which is fundamental in clinical treatments and pharmacological trials.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40035-023-00367-9.