The spontaneous changes in brain activity in patients with diabetic optic neuropathy using steady-state fMRI. The fractional amplitude of the low-frequency fluctuation method was applied to evaluate neural activity changes. The Hospital Anxiety and Depression Scale was used to assess the anxiety and depression status of participants. The independent sample t-test and chi-squared test were applied to analyze the demographics of diabetic optic neuropathy patients and healthy controls. Receiver operating characteristic curves were applied to analyze the variation in mean fractional amplitude of low-frequency fluctuation values between diabetic optic neuropathy patients and healthy controls. Pearson’s correlation analysis analyzed the relationships between the fractional amplitude of low-frequency fluctuation values of brain regions and clinical behaviors in the diabetic optic neuropathy group. The fractional amplitude of low-frequency fluctuation value of diabetic optic neuropathy patients was significantly higher than healthy controls in the right precentral gyrus. However, the fractional amplitude of low-frequency fluctuation values in the right anterior cingulate gyrus and left middle cingulate gyrus were markedly decreased in diabetic optic neuropathy patients. The area under the curve of receiver operating characteristics for each brain region showed high accuracy. Pearson’s correlation analysis showed that fractional amplitude of low-frequency fluctuation values of the right anterior cingulate gyrus and left middle cingulate gyrus was negatively correlated with Hospital Anxiety and Depression Scale scores. The fractional amplitude of low-frequency fluctuation values of the left middle cingulate gyrus was negatively correlated with diabetic optic neuropathy disease duration. In conclusion, we found abnormal spontaneous brain activities in regions related to cognitive and emotional dysfunction, eye movement disorder, and vision loss in patients with diabetic optic neuropathy. These results may indicate the underlying neuropathological mechanisms of diabetic optic neuropathy and show that fractional amplitude of low-frequency fluctuation may be an effective method to distinguish patients with diabetic optic neuropathy from healthy individuals.