Objective: It was the aim of this study to compare the diagnostic efficiency of anti-aquaporin 4 (AQP4) antibody detection between serum and cerebrospinal fluid (CSF) samples in Chinese patients with central nervous system demyelinating diseases. Methods: Anti-AQP4 antibodies were detected by a cell-based assay. We calculated the sensitivity, specificity and coherence in 118 patients with neuromyelitis optica (NMO, n = 39), multiple sclerosis (n = 34), longitudinally extensive transverse myelitis (LETM, n = 22), optic neuritis (ON, n = 6), opticospinal multiple sclerosis (n = 8) and acute partial transverse myelitis (n = 9). Results: Forty-four serum samples (33.8%) were positive for anti-AQP4 antibodies. Anti-AQP4 antibody seropositivity was 76.9, 59.1 and 16.7% in patients with NMO, LETM and ON, respectively. Sixty-five CSF samples (50%) were positive for anti-AQP4 antibodies. Anti-AQP4 antibody positivity was 87.1, 81.8, 83.3, 62.5 and 11.8% in patients with NMO, LETM, ON, opticospinal multiple sclerosis and multiple sclerosis, respectively. The ĸ value of the coherence test was 0.585 (p < 0.0001) between the two types of samples. The antibody positivity rate was significantly different between the two body fluids (p = 0.0008, McNemar test). The sensitivity and specificity were 74.3 and 100% in serum, 85.7 and 88.2% in CSF, and 94.3 and 88.2% for serum and CSF combined, respectively. Conclusion: The sensitivity of anti-AQP4 antibodies in the CSF was higher than that in the serum, and their combined use is helpful in diagnosing Chinese patients with NMO.