Neuromyelitis Optica (NMO) is a neuroinflammatory disease marked by severe attacks on the optic nerves and spinal cord. While it is established that NMO affects brain function, the detailed progression of these impacts over time remains poorly investigated. The objective of this study is to investigate spontaneous temporal changes in brain function in patients with NMO and to explore the associations between these changes and clinical assessment. This longitudinal study recruited 31 non-relapsing patients with NMO, for whom resting-state functional MRI (rs-fMRI) data were collected at baseline and follow-up. Besides, 20 age- and sex-matched healthy controls (HCs) were included and assessed only at baseline. Neural activity was quantitatively assessed using the amplitude of low-frequency fluctuations (ALFF). We analyzed differences in brain function between NMO patients and HCs, as well as changes within the patient group over time. Additionally, we examined correlations between changes in ALFF and clinical outcomes, including the Expanded Disability Status Scale (EDSS) and visual acuity. At baseline, significant ALFF reductions were observed in NMO patients, particularly in the bilateral paracentral lobules, posterior central gyrus, inferior temporal gyri, lingual gyri, right precentral gyrus, middle and inferior occipital gyri, and fusiform gyrus. Over time, affected areas expanded, particularly in the occipital and temporal lobes, and initially unaffected regions like the superior temporal gyrus and calcarine areas showed significant reductions at follow-up. Initially, NMO patients exhibited higher ALFF in the cerebellum, bilateral pons, parahippocampus, thalamus, posterior and anterior cingulate cortex, and precuneus. However, by the follow-up, elevated ALFF persisted only in the medial superior frontal gyrus. During follow-up, progressive decreases in ALFF were specifically noted in the right lingual gyrus, calcarine, fusiform gyrus, precuneus, and parahippocampus within the NMO patient group. Significant correlations were identified between improvements in EDSS scores and increases in ALFF in the paracentral lobule, precentral, and postcentral gyri. Additionally, enhancements in visual acuity were linked to increased ALFF in the medial superior frontal gyrus. Rs-fMRI reveals progressive brain function declines in NMO, evidenced by decreasing ALFF in key sensory and motor areas, alongside occasional compensatory increases observed. Strong correlations between these changes and clinical measures like EDSS scores and visual acuity highlight ALFF's value as a biomarker for monitoring disease spontaneous progression and assessing treatment impacts.