Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system affecting over 2.8 million people worldwide. Early diagnosis is critical to initiate treatment before irreversible neurodegeneration but current diagnostic methods are non-specific, invasive and costly. There is an urgent need for a specific blood-based diagnostic test. Epstein-Barr virus (EBV) infection has emerged as a necessary precursor of MS, suggesting this link could be leveraged to develop a diagnostic test for MS onset. We hypothesise that MS patients harbour distinct autoantibody signatures in CSF and plasma that correlate with EBV-specific antibody responses, and that this integrated EBV/autoantibody signature would be a predictive blood-based biomarker for MS onset. We profiled EBV antibody reactivity across the entire EBV proteome in plasma and CSF samples from subjects with clinically confirmed MS at disease onset, using an EBV proteome microarray spanning all lifecycle stages and species (>200 proteins). We profiled human autoantibody reactivity of matched CSF and plasma samples using the HuProt 4.0 human proteome microarray (>21,000 proteins). Integrative analyses defined disease-associated autoantibody repertoire and evaluated correlations between human and EBV antibody responses. A distinct MS-specific EBV antibody signature associated with MS onset was identified in plasma. A core EBV antibody signature was present in matched CSF and plasma samples, indicating that systemic EBV-directed immunity is reflected within the CNS. Complementary human proteome profiling revealed both shared and CSF-enriched autoantibody responses in MS compared with Other Neurological Disease controls. Cross-platform integration to define candidate autoantigens and pathways linking viral exposure to CNS-targeted autoimmunity is in progress. This study is the first integrative proteome-wide characterisation of EBV- and human-specific antibody responses in paired CSF and plasma from MS patients. By defining disease-specific immune signatures, we provide novel insights to support development of a blood-based biomarker for early and accurate disease detection of MS.