Anti‐neurofascin‐155 (NF155) antibodies have been observed in two cases with neuromyelitis optica spectrum disorders (NMOSD). This study investigated the prevalence of anti‐NF155 antibodies in patients with NMOSD and the clinical features of anti‐NF155 antibody‐positive patients. Sera from 129 patients with NMOSD were screened with anti‐NF155 antibodies by cell‐based assay (CBA) and re‐examined using immunostaining of teased mouse sciatic nerve fibres. Fifty‐six patients with multiple sclerosis (MS) and 50 healthy controls (HC) were also enrolled for detecting anti‐NF155 antibodies. A total of 12.40% (16 of 129) of patients with NMOSD were positive for anti‐NF155 antibodies confirmed by both CBA and immunostaining. Immunoglobulin (Ig) G1 was the predominant subclass. However, none of 56 MS patients or 50 HC were positive for anti‐NF155 antibodies. Anti‐NF155 antibody‐positive NMOSD patients had a higher proportion of co‐existing with autoimmune diseases (p < 0.001) and higher positive rates of serum non‐organ‐specific autoantibodies, including anti‐SSA antibodies (p < 0.001), anti‐SSB antibodies (p = 0.008), anti‐Ro‐52 antibodies (p < 0.001) and rheumatoid factor (p < 0.001). Five anti‐NF155 antibody‐positive NMOSD patients who took part in the nerve conduction study showed mildly abnormal results. Differences in some nerve conduction study parameters were observed between anti‐NF155 antibody‐positive and negative patients. Anti‐NF155 antibodies occurred in a small proportion of NMOSD patients. Anti‐NF155 antibody‐positive NMOSD patients tended to co‐exist with autoimmune diseases.