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Abstract Background Glial fibrillary acidic protein (GFAP) is an intermediate filament protein specifically present in astrocytes in the central nervous system. Elevated levels of GFAP in the cerebrospinal fluid (CSF) and blood of patients have been reported to be correlated to the pathology of several neurological disorders, the occurrence of traumatic brain injury, and the severity of the neurological deficit and extent of brain damage from ischemic stroke, brain tumors, etc. In addition, when considering the clinical usefulness of GFAP or also the other novel neurological biomarkers, it has also been reported that the combination of those biomarkers may improve their diagnostic performances. Despite their potential utility, availability of patient specimens for use in studying these biomarkers is quite limited. Therefore, there is an increasing demand for the development of new measurement systems that can provide highly accurate results with a smaller sample volumes. To address this demand, we have developed a novel ultrasensitive blood-based GFAP immunoassay utilizing single-molecule counting technology (Fluxus, Inc., USA) and we present its key performance characteristics. Methods An immunoassay for GFAP was developed based on two-step sandwich method using our in-house developed monoclonal antibodies immobilized to magnetic particle for capture and fluorescence labeled for detection. Fluorescence labeled reporters were released after immunoreaction and injected into the detector device for single-molecule counting. We evaluated its analytical sensitivity, method comparison, and clinical performance. The limits of detection (LoD) and quantification (LoQ) were calculated with the measurement of EDTA-plasma based low concentration GFAP samples for the analytical sensitivity. The method comparison was performed against Lumipulse® G GFAP (Fujirebio Inc., Japan) as the other established GFAP assay and the correlation coefficient and the regression slope were determined by the Passing-Bablok regression analysis. Commercially obtained Alzheimer’s donor (AD) samples and healthy donor (HD) samples were measured for the evaluation of clinical performance. Results LoD and LoQ was calculated in 0.47 and 2.11 pg/mL, respectively. The correlation coefficient against Lumipulse® G GFAP as the other established GFAP assay resulted in 1.00 and indicated good results for the regression slope as well. The evaluation of clinical samples was consistent with the previous reports, showing reasonable measurement levels and a tendency for higher values in AD samples compared to HD samples, with a significant difference (p-value less than 0.001). Conclusion The assay demonstrated excellent analytical sensitivity and potential clinical utility in AD based on blood samples. Our fluorescence immunoassay on the Fluxus single-molecule counting platform provides ultrasensitive detection of blood-based GFAP, allowing the evaluation of specimens using very small volumes. This capability can help accelerate the further research into GFAP’s roles in neurological disorders and aid clinical management.