Background: NF-light, Tau, GFAP and UCH-L1 are emerging neurology biomarkers relevant for neurodegeneration and traumatic brain injury. Simultaneous measurement of these biomarkers in blood and CSF of both healthy and diseased individuals is of great interest. Methods: A fully-automated multiplexed digital immunoassay for these 4 neurology biomarkers was developed based on Single Molecule Array (Simoa) technology with ultra-sensitivity. The assay reagents were designed for use on the Simoa HD-1 Analyzer. Firstly, an immunocomplex was formed by incubating calibrators or samples with capture antibody-coated paramagnetic beads and biotinylated detector antibody. Then, the immunocomplex was labeled with streptavidin-β-galactosidase conjugate followed by incubation with the fluorogenic substrate resorufin-β-D-galactopyranoside. Beads were loaded into a microwell array to produce single beaded wells and digital signals. The individual assays in the plex were evaluated for sensitivity, linearity, spike/recovery, plex-cross reactivity and detectability in serum, plasma, and CSF from healthy donors. 207 plasma samples from individuals with high or low blast exposure were tested using the 4-plex assay. Results: The limits of detection were 0.087pg/mL (NF-light), 0.010pg/mL (Tau), 0.229pg/mL (GFAP), 1.357pg/mL (UCH-L1). Spike/recovery and dilution linearity were all within 80-120%. There was minimal cross reactivity between individual assays. Plasma, serum and CSF samples from healthy donors were tested to evaluate the multiplex. The levels of all four analytes from the plexed assays showed good correlation with the levels from single-plex assays. Blast sample test with 4-plex showed differentiation between mild (exposure of 2psi or lower) and acute (exposure of 8psi or higher) TBI patients. Conclusions: The Simoa neurology 4-plex assay is able to reliably measure four biomarkers simultaneously in plasma, serum and CSF samples from healthy individuals. It provides a new tool for the quantification of NF-light, Tau, GFAP and UCH-L1 in both normal and disease samples, facilitating the investigation of traumatic brain injury and neurodegenerative diseases.