Accurate diagnosis of Alzheimer’s disease (AD) in its earliest stage can prevent the disease and delay the onset of symptoms by maintaining a healthy lifestyle and controlling modifiable risk factors. Therefore, more sensitive, relatively inexpensive, non-invasive, and simple screening tools are required for the early diagnosis and progression monitoring of AD. Here, we designed a self assembled nanoparticle-mediated amplified fluorogenic immunoassay (SNAFIA) consisting of magnetic and fluorophore-loaded polymeric nanoparticles. Multiple simultaneous captures and magnetic separation of biomarkers and target-specific fluorophore emission induced a one-tomulti signal, resulting in a low detection limit (236 aM), good reliability (R2 = 0.991), and wide analytical range (0.320–1000 fM) for APOE and CAP1 biomarkers in human serum and tear fluid. More importantly, SNAFIA successfully differentiated AD patients from healthy controls with 90% sensitivity and 100% specificity in less than 1 h in 39 clinical tear samples. As an easily accessible diagnostic tool with fast and accurate results, SNAFIA can help diagnose AD early to aid in long-term care planning, increase the efficiency of clinical trials, and dramatically accelerate therapeutic development.