INTRODUCTION: Blood-based biomarkers offer scalable, non-invasive tools for Alzheimer's disease (AD) detection. We investigated the performance of plasma biomarkers associated with AD on the automated Beckman Coulter Access DxI 9000 analyzer. METHODS: This cross-sectional study included 262 individuals from across the AD continuum. Plasma phosphorylated tau at threonine 217 (p-tau217), amyloid beta (Aβ)42, and their ratio were measured. Diagnostic accuracy for amyloid positron emission tomography (PET) positivity (Centiloid > 20), using a dual cutoff approach, was assessed via receiver operative characteristic curve. Associations with tau PET (n = 76) were also assessed. RESULTS: The p-tau217/Aβ42 ratio showed the highest diagnostic accuracy for amyloid PET positivity (area under curve = 0.943) and the smallest indeterminate zone (8.0%). It correlated strongly and consistently with tau PET across Braak stages and with AD-related cortical atrophy. DISCUSSION: The p-tau217/Aβ42 ratio was the most reliable plasma biomarker, closely tracking tau PET. It has potentials for clinical use in diagnosis and treatment monitoring. HIGHLIGHTS: This is the first validation of the Beckman Coulter plasma immunoassay. The plasma phosphorylated tau at threonine 217 amyloid beta 42 ratio showed the highest accuracy across the full Alzheimer's disease (AD) spectrum. Plasma biomarkers correlated with tau positron emission tomography and AD-related brain atrophy. Glial fibrillary acidic protein offered complementary value reflecting astrocytic activation.