Background: Neovascular glaucoma (NVG) is a serious eye disease that causes irreversible damage to the eye. It can significantly increase intraocular pressure and cause severe pain, as well as abnormal activity in the cortical and pre-cortical visual systems. However, there are few studies in this area. This trial assessed the altered regional brain activity in patients with NVG using the percentage of fluctuation amplitude (PerAF) method.Methods: Resting-state functional MRI (rs-fMRI) scans were conducted in 18 individuals with NVG and 18 healthy controls (HCs), matched for education level, gender, and age. The PerAF method was applied to assess brain activity. Mean PerAF values of brain regions in NVG and HCs were compared using receiver operating characteristic (ROC) curves.Results: Lower PerAF values were found in the NVG group than in controls in the right anterior cingulate and paracingulate gyri (ACG.R), right superior occipital gyrus (SOG.R) and left superior frontal gyrus (orbital part) (ORBsup.L) (p < 0.001). In contrast, PerAF value was higher in NVG patients than in controls in the left inferior temporal gyrus (ITG.L) (p < 0.001). The hospital anxiety and depression scale (HADS) and visual analog score (VAS) were significantly and positively correlated with PerAF in ITG.L (r = 0.9331, p < 0.0001; and r = 0.7816, p = 0.0001, respectively).Conclusion: Abnormal activity in the patient’s brain regions further confirms that the NVG affects the entire brain, not just the visual pathways and posterior retinal mechanisms (including the hypothalamic lateral geniculate nucleus and the primary visual cortex). This strengthens our understanding of the NVG and provides potential diagnostic and therapeutic support for patients who are difficult to diagnose and treat early.