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Objective To evaluate the diagnostic value of plasma β-amyloid (Aβ) seeding activity measured using a newly developed instrument to distinguish Alzheimer’s disease (AD) from other forms of dementia. Methods Seventy-nine AD patients, 64 non-AD dementia (NADD) patients, and 75 cognitively normal (NC) subjects were recruited in the study. To measure the levels of Aβ seeding activity in the plasma samples, we have developed an AD-seeds protein analyzer. We used receiver operating characteristic (ROC) curves to quantify the ability of plasma Aβ seeding activity to distinguish between AD and NADD or NC individuals. Spearman’s correlation was used to examine the associations between plasma Aβ seeding activity and global cognitive function or conventional AD biomarkers. Results The Aβ seeding activities were 0.83 (0.58–1.16) A.U. in AD, 0.42 (0.04–0.74) A.U. in NADD and 0.42 (0.09–0.69) A.U. in NC, respectively. The Aβ seeding activity was able to identify AD patients and distinguish them from NC or NADD with high accuracy (AUC = 0.85–0.86). In addition, the plasma Aβ seeding activity showed a strong correlation with cognitive performance (mini-mental state examination,  r  = − 0.188; Montreal cognitive assessment,  r  = − 0.189; clinical dementia rating,  r  = 0.205) and conventional biomarkers (cerebrospinal fluid [CSF] Aβ42/40, r = -0.227; CSF T-tau/Aβ42,  r  = 0.239; CSF P-tau/Aβ42,  r  = 0.259). Conclusion Our results confirmed that plasma Aβ seeding activity is an antibody-free and low-cost biomarker for the diagnosis of AD. Trial registration Trial registration number  NCT04850053

Detection of plasma Aβ seeding activity by a newly developed analyzer for diagnosis of Alzheimer’s disease