Abstract INTRODUCTION This study aimed to investigate the differential roles of various plasma biomarkers in a stepwise diagnostic strategy for Alzheimer's disease (AD). METHODS A total of 2984 participants, including 666 cognitively unimpaired (CU), 2032 with Alzheimer's clinical syndrome (ACS), and 286 non‐ACS individuals, were recruited. Plasma amyloid beta (Aβ) 42/40, four phosphorylated tau (p‐tau) epitopes, glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL) levels were measured using immunoassays. RESULTS NfL demonstrated fair to excellent accuracy in differentiating non‐ACS from CU groups (area under the curve [AUC], 0.79 to 0.94). p‐tau217 had the highest accuracy for identifying Aβ (AUC 0.94) and tau positron emission tomography status (AUC 0.91). In the ACS group, p‐tau217 was the strongest predictor of cognitive decline (p < .001). DISCUSSION NfL may serve as a useful screening tool, while p‐tau217 is particularly valuable for confirming AD pathology and prognosis. Highlights Plasma NfL could screen for cognitive impairment. p‐tau217 reliably detects AD pathology, regardless of diagnosis. p‐tau217 and GFAP predict prognosis in ACS. Each plasma biomarker plays a distinct role in stepwise AD diagnostics.