Background: Neuromyelitis optica spectrum disorder (NMOSD) refers to a range of autoimmune inflammatory demyelinating diseases affecting the optic nerves, spinal cord, and periependymal regions of the brain. Classical NMOSD is characterized by the presentation of autoantibodies against the water channel aquaporin-4 (AQP4). However, a subset of patients fulfilling the clinical criteria for NMOSD is negative for AQP4-IgG but positive for autoantibodies against myelin oligodendrocyte glycoprotein (MOG); these patients are associated with different clinical manifestations and pathogenesis. Methods: Patients who received a first diagnosis of NMOSD were reviewed retrospectively between April 2015 and December 2018. Patients were classified according to the presence of AQP4-IgG and MOG-IgG in serum and/ or cerebrospinal fluid. Clinical characteristics, magnetic resonance imaging findings, disease severity, and serum C3 and C4 levels at the first episode were compared between the groups. Results: The NMOSD patients with AQP4-IgG and MOG-IgG demonstrated specific, differential clinical features. The AQP4-IgG group featured more women, the presentation of transverse myelitis attacks and simultaneous occurrence of optic neuritis and transverse myelitis were more common, and intrathecal synthesis was more evident. The MOG-IgG group featured younger patients, more acute disseminated encephalomyelitis (ADEM) or ADEM-like attacks, more frequent cerebrospinal fluid pleocytosis, and a better overall outcome. C3 levels were significantly lower in AQP4-IgG-positive patients and higher in MOG-IgG-positive patients relative to healthy controls. C4 levels were significantly lower in the AQP4-IgG-positive NMOSD group when compared to both MOG-IgG-positive patients and controls. C3 and C4 were then combined in a receiver operating characteristic model. The area under the curve of the model was calculated to differentiate the AQP4-IgG-positive group from the MOG-IgG-positive group was 0.787, which was considered moderately predictive. Conclusion: The combination of C3 and C4 could assist in the differential diagnosis of AQP4-IgG-positive NMOSD from MOG-IgG-positive NMOSD.