BACKGROUND: We aimed to compare the analytical performance of four assays, for measuring β-amyloid 1-42 (Aβ1-42), β-amyloid 1-40 (Aβ1-40), total tau, and p-tau181 in cerebrospinal fluid (CSF) and evaluate the clinical performance for the diagnosis of Alzheimer's disease (AD). METHODS: The measured concentrations and analytical performance, including precision, linearity, and accuracy of four assays were compared. The discriminative accuracy for amyloid positron emission tomography (PET) status based on different assays was evaluated. RESULTS: The measurements of the four biomarkers based on the four assays demonstrated favorable agreement, while still significantly different. For the discrimination of PET status, the ratio of Aβ1-42/p-tau181 showed better diagnostic performance than other biomarkers, with liquid chromatography tandem-mass spectrometry (LC-MS/MS) and Lumipulse G assays performing better when combining all biomarkers for each assay. CONCLUSIONS: It is crucial to promote the harmonization and standardization of pre-analytical and measurement procedures to achieve consistent and comparable results in different assays and laboratories.