Aim To explore the altered spontaneous cerebral activity patterns and impaired functional regions in patients with diabetic retinopathy (DR) using resting-state functional magnetic resonance imaging (rs-fMRI) based on the amplitude of low-frequency fluctuations (ALFF) algorithm. Materials and methods Twenty-one patients with DR (mean age, 54.9±9.9 years; 11 females) and 17 healthy control subjects (54.8±5.7 years; 9 females) were prospectively studied. The DR patients underwent laboratory tests. All individuals underwent a neuropsychological test. The differences in the ALFF values between the two groups were compared. The relationships between ALFF values and clinical measurements were analysed using a multiple-factor analysis. Results Compared to the controls, the DR group showed significantly increased ALFF values in the bilateral occipital gyrus, right lingual gyrus, and precuneus, and decreased values in the right posterior/anterior cerebellar lobe and the parahippocampal, fusiform, superior temporal, inferior parietal, and angular gyrus. Furthermore, the Montreal Cognitive Assessment (MoCA) scores were negatively correlated with decreased ALFF values in the right occipital lobe of the DR group, while increased ALFF values in the right precuneus and lingual gyrus were found to be positively correlated with glycosylated haemoglobin (HbA1c) levels. Conclusions Patients with DR showed spontaneous cerebral activity abnormalities in many cerebral regions that were associated with cognitive impairments. HbA1c levels altered spontaneous cerebral activity in DR patients.