The objective of this study is to observe the visual fatigue caused by watching 3DTV using the method of functional magnetic resonance imaging (fMRI). The data of fMRI during three kinds of visual stimulation tasks were obtained from twenty subjects. At first, blood-oxygen-level dependent (BOLD) signal changes during stimuli of checkerboard task were compared before and after one-hour watching 3D/2DTV, and subjective evaluation was conducted based on the questionnaire simultaneously. Then 3D and 2D images were used to stimulate healthy individuals to measure brain activities that correlated with stereoscopic vision. Finally, the relationship between front or back depth of field images and visual fatigue was investigated. The results reveal that the 3D group shows more significant differences of brain activities in BA8, BA17, BA18 and BA19 than the 2D group during the checkerboard stimulation. BA5, BA6, BA7 and BA8 were testified to have close relationship with stereoscopic perception via the 2D/3D images stimulation. Furthermore, the front depth of field image was proven to impose a more serious impact on visual fatigue than the back one. These conclusions are useful for healthy and reasonable 3DTV watching as well as properly designing of 3D scenes.