ObjectiveThe objective of this study is to investigate the presence of astrocyte antibodies in patients, excluding aquaporin‐4 or glial fibrillary acidic protein (GFAP) antibodies, while evaluating associated biomarkers and pathologies.MethodsPatient serum and cerebrospinal fluid (CSF) were tested for antibodies using tissue‐ and cell‐based assays. Neurofilament light chain (NFL) and GFAP in the CSF were detected using single‐molecule array (SIMOA).Results116 patients accepted SIMOA. Fifteen functional neurological disorders patients without antibodies were designated as controls. Thirty‐five patients were positive for astrocyte antibodies (Anti‐GFAP: 7; Anti‐AQP4: 7; unknown antibodies: 21, designed as the double‐negative group, DNAP). The most frequent phenotype of DNAP was encephalitis (42.9%), followed by myelitis (23.8%), movement disorders (19.0%), and amyotrophic lateral sclerosis‐like (ALS‐like) disease (14.2%). The levels of CSF GFAP and NFL in DNAP were higher than in the control (GFAP: 1967.29 [776.60–13214.47] vs 475.38 [16.80–943.60] pg/mL, p < 0.001; NFL: 549.11 [162.08–2462.61] vs 214.18 [81.60–349.60] pg/mL, p = 0.002). GFAP levels decreased in DNAP (n = 5) after immunotherapy (2446.75 [1583.45–6277.33] vs 1380.46 [272.16–2005.80] pg/mL, p = 0.043), while there was no difference in NFL levels (2273.78 [162.08–2462.61] vs 890.42 [645.06–3168.06] pg/mL, p = 0.893). Two brain biopsy patterns were observed: one exhibited prominent tissue proliferation and hypertrophic astrocytes, with local loss of astrocytes, while the other showed severe astrocyte depletion with loss of neurofilaments around the vessels. Eighteen patients received immunotherapy, and improved except one with ALS‐like symptoms. We identified anti‐vimentin in this patient.DiscussionThere are unidentified astrocyte antibodies. The manifestations of double‐negativity are heterogeneous; nevertheless, the pathology and biomarkers remain consistent with astrocytopathy. Immunotherapy is effective.