Neuromyelitis optica (NMO) is a devastating neurologic disease characterized by severe optic neuritis and transverse myelitis. Recently, its disease-specific serum autoantibody, NMO-IgG, was discovered with indirect immunofluorescence. However, the substrates of the immunofluorescence assay were not human but mouse brain tissues, which could influence the sensitivity and specificity of the antibody. The target antigen of NMO-IgG was recently identified as aquaporin-4 (AQP4) water channel protein, which is mainly expressed in brain and spinal cord. In the present study, we have established human cell lines that stably express human AQP4 and used these cells to detect and titrate anti-AQP4 antibody present in the sera of patients with NMO by immunofluorescence assay. The results were compared with those of the original NMO-IgG assay. We tested the sera from 10 patients with NMO, 10 with MS and five with other neurological disorders. Among the patients with NMO, six were NMO-IgG-positive. However, using the new anti-AQP4 antibody assay, we showed that eight patients with NMO including the six NMO-IgG-positives were positive for anti-AQP4 antibody. The staining pattern of AQP4-expressing cells treated with each serum of these eight NMO patients corresponded to that with a commercially available anti-AQP4 antibody. The antibody titer (maximum serum dilution for positive staining) ranged from 64× to 16,384×. The serum dilution titers were reproducible in blinded studies. In contrast, the patients with MS or other neurological disorders showed negative for anti-AQP4 antibody. Thus, the newly developed anti-AQP4 antibody assay appears to have a higher sensitivity for NMO than the original NMO-IgG assay and is expected to be useful for the diagnosis of NMO.