Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Although various viruses have been proposed to contribute to MS pathology, the etiology of MS remains unknown. Since intrathecal antibody synthesis is well documented in chronic viral infection and neuroinflammatory diseases, we hypothesized whether the patterns of antigen-specific antibody responses associated with various viral exposures may define patients with CNS chronic immune dysregulation. The pan-viral antibody profiling in cerebrospinal fluid (CSF) and serum of patients with MS showed significant differences from those in healthy volunteers and a pattern of antibody responses against multiple viruses, including the previously identified Epstein-Barr virus. These findings demonstrate that virus-specific antibody signatures might be able to reflect disease-associated inflammatory milieu in CSF of subjects with neuroinflammatory diseases.