Objective: To investigate alterations in brain neural networks using resting-state functional magnetic resonance imaging (rs-fMRI) and regional homogeneity (ReHo) analysis in patients with chronic fatigue syndrome (CFS) and comorbid anxiety and depression, and to explore implications for physical performance, rehabilitation, and sports participation. Methods: Sixty participants were recruited, including 30 patients with CFS and anxiety-depression comorbidity and 30 healthy controls matched for age, sex, and lifestyle. rs-fMRI was conducted to assess neural connectivity changes, and ReHo analysis was applied to identify alterations in brain activity. Correlation analyses were performed to examine the relationship between ReHo values and clinical features in affected brain regions. The severity of CFS with anxiety and depression was assessed using validated scales, including the Multidimensional Fatigue Inventory (MFI-20), Self-Rating Depression Scale (SDS), Self-Rating Anxiety Scale (SAS), and Pittsburgh Sleep Quality Index (PSQI). Results: Patients with CFS and anxiety-depression comorbidity exhibited significantly different scores on clinical scales compared to healthy controls (p < 0.05). Brain regions with increased ReHo values in the patient group included the left inferior lobe, interhemispheric area, right occipital lobe, and thalamus. Decreased ReHo values were observed in the left and right middle temporal gyri, right posterior central gyrus, right middle orbital frontal gyrus, right paracentral lobule, and right fusiform gyrus. Correlation analysis revealed positive associations between clinical scales and ReHo values in the thalamus, interhemispheric area, and right middle orbital frontal gyrus. Negative correlations were found between ReHo values and the right occipital lobe, left inferior cerebral lobe, and fusiform gyrus. Conclusion: Altered regional homogeneity in specific brain areas in patients with CFS and comorbid anxiety and depression suggests imbalances in neural network excitation and inhibition. These findings provide insights into the neurological basis of fatigue and emotional disturbances in this population and highlight potential targets for therapeutic interventions. Understanding these brain network changes can inform tailored rehabilitation strategies, focusing on improving cognitive function, physical performance, and participation in sports or exercise-based programs. This study underscores the importance of addressing neuropsychological factors in comprehensive rehabilitation for CFS patients.