Objective: This paper studies the fatigue caused by prolonged watching of three-dimensional television (3DTV) by applying the amplitude of low frequency fluctuation (ALFF) and the community partitioning algorithm in brain functional network techniques to resting-state functional magnetic resonance imaging (fMRI). In this study, two specific frequency bands (Slow5: 0.01-0.027 Hz and Slow4: 0.027-0.073 Hz) were analyzed to investigate the change of brain spontaneous activity after one-hour watching of 3DTV. Meanwhile, the changes of brain modular organization were analyzed to discuss the physiological mechanism of brain fatigue during long period of watching 3DTV. Methods: Valid data of resting state fMRI from 20 dextromanual subjects were adopted before and after 1 h watching of 3DTV/2DTV. Then, ALFF was processed using SPM8 and REST software to analyze the variation of resting-state fMRI induced by watching 2D/3DTV. Community partitioning algorithm was used to evaluate the change of brain modular organization. Results: While watching 2DTV does not significantly change the brain spontaneous activity, watching 3DTV makes significant differences on middle frontal gyrus (BA9/BA10), right inferior occipital gyrus (BA18/19), right inferior temporal gyrus (BA20/37) and cingulate cortex (BA32). The result shows that dorsal stream was activated after watching 3DTV through analyzed modularity and community, which verifies that watching 3DTV requires the brain visual pathway to process stereoscopic display. Meanwhile, the result further reflects that the neural circuit, including visual conducting pathway, temporal lobe and basal ganglia loop, is closely related to brain fatigue caused by long period of watching 3DTV. The study has shown the potential risk of 3DTV to viewers' health, especially on vision and recognition functions. The basal ganglia neurons play crucial roles in the physiological mechanism of brain fatigue. Therefore, prolonged 3DTV watching should be avoided particularly for physically developing teenagers. This research suggested that the ALFF technique and brain modular organization is able to reveal intrinsic changes caused by visual fatigue and provided a foundation for 3DTV fatigue assessment.