Fluorescent nanodiamond (FND) has been regarded as a superior alternative reporter for lateral flow immunoassays. The negatively charged nitrogen-vacancy (NV−) center in FND is a point defect with unique magneto-optical properties, giving outstanding characteristics to detect biomarkers of diseases, including Alzheimer’s disease (AD). AD is the most common form of dementia, which might ultimately result in complete brain failure and, eventually, death. The two most commonly used techniques to detect brain changes caused by AD are brain imaging and lumbar puncture. However, brain imaging and lumbar puncture are costly and time-consuming. Thus, we have developed Spin-Enhanced Lateral Flow Immunoassay (SELFIA) for in vitro AD diagnostics utilizing the electron spin properties of the NV−-centers in FND to achieve background-free ultrasensitive detection. To detect the pTau protein (a critical AD biomarker), we first conjugated FND to anti-pTau antibodies through non-covalent conjugation. We employed a sandwich assay to enhance sensitivity and specificity by immobilizing capture antibodies on the membrane so that antibody-antigen–antibody immunocomplexes would be formed. Our FND-based SELFIA only requires approximately 30 min to reach a detection limit of 7 pg/mL, where the threshold for positivity was 101.95 pg/mL for cerebrospinal fluid pTau217. This is the first study that utilized FND-based SELFIA for AD detection. The current SELFIA platform is rapid, easy to use, and affordable. This study not only represents a significant innovation but also opens new avenues for early diagnosis and treatment strategies.