Objective <p>Serum glial fibrillary acidic protein (sGFAP) and neurofilament light chain (sNfL) levels are potential biomarkers for disease activity and disability in Neuromyelitis Optica Spectrum Disorder (NMOSD), respectively; however, their clinical applications in NMOSD remain unclear. Therefore, this study aims to investigate the correlation between sGFAP and sNfL levels and the activity, severity of attacks, disability in patients with NMOSD.</p> Methods <p>SGFAP and sNfL levels were measured in healthy controls (HCs) (<i>n</i> = 20) and patients with NMOSD (<i>n</i> = 86; 50 during attack and 50 in remission) using ultrasensitive single-molecule array assays. The relationships between the two biomarkers and different stages of NMOSD, the severe attacks, the Expanded Disability Status Scale (EDSS) scores, and the prognosis were analyzed.</p> Results <p>SNfL and sGFAP levels were significantly higher in patients with NMOSD than in HCs in the acute and remission phases (<i>p</i> &lt; 0.05). The sGFAP level was higher in the acute phase than in the remission phase (<i>P</i> &lt; 0.001). SNfL and sGFAP levels were higher in the severe myelitis attack group than in the severe optic neuritis attack group (<i>p</i> &lt; 0.05). The EDSS ≥ 6 NMOSD group had significantly higher sNfL and sGFAP levels than the EDSS &lt; 6 group (<i>p</i> &lt; 0.05). SNfL and sGFAP levels in acute and remission stages increased with increasing EDSS scores (<i>p</i> &lt; 0.05). SGFAP could discriminate patients with NMOSD with relapse from those under remission (<i>p</i> &lt; 0.001).</p> Conclusion <p>sNfL and sGFAP may serve as biomarkers for NMOSD severity, activity, and motor disability.</p>