Objective: To develop a sensitive and reliable diagnostic approach for optic neuromyelitis (ONM), an overexpressing cell line capable of detecting aquaporin 4 (AQP4) antibodies was established. Subsequently, immunofluorescence was employed to detect AQP4 antibodies in serum and cerebrospinal fluid (CSF) samples from patients. Additionally, the clinical utility of AQP4 antibodies in the detection of ONM patients was analyzed. Methods: An aquaporin 4 expression plasmid was constructed and transfected into cell lines. Subsequently, indirect immunofluorescence was utilized to detect anti-AQP4 antibodies in serum and cerebrospinal fluid samples. Results: The indirect immunofluorescence detection system exhibited high sensitivity in the detection of 2241 clinical samples, with a positive rate of 16.8%. The positive proportion was in line with epidemiological data, and the positive situation was consistent with clinical symptoms. Conclusion: The engineered cell line exhibits superior detection performance for identifying antibodies present in serum and cerebrospinal fluid samples. The capability to detect anti-aquaporin 4 antibodies is pivotal for improving the efficiency of screening and diagnosing neuromyelitis optica, thereby possessing considerable potential for clinical application.