Visual fatigue is considered to be an indicator responsible for a user’s visual experience of watching stereoscopic display, and requires an efficient evaluation method. This article compares the visual fatigue based on electrocardiograph (ECG) analysis on 3D real objects versus stereoscopic TV, involving a magic cube based task in which participants are required to view magic cubes in the real environment and virtual images on stereoscopic TV. The results of ECG analysis show a significant increase of approximate entropy (ApEn) and stable changes of 15 other parameters. The ApEn variation shows that the visual fatigue when viewing stereoscopic TV is more severe than that when viewing magic cubes in the real environment after a 20-minute viewing session. In addition, the results for critical flicker frequency are consistent with those of ApEn, and the results of a subjective questionnaire show that the proposed experiment has induced visual fatigue. The results for correct rate also show that the visual fatigue when viewing virtual images on stereoscopic TV is more severe than that when viewing magic cubes in the real environment after a six-minute viewing session. It can be concluded that the visual fatigue when viewing stereoscopic TV appears earlier than that when viewing 3D real objects and is more severe than that when viewing 3D real objects. However, severe visual fatigue is induced both by viewing 3D real objects and by viewing stereoscopic TV after a 25-minute viewing session, and it does not show significant difference between viewing 3D real objects and viewing stereoscopic TV, which proves that the findings in this article may have potential impacts for the improvement of visual fatigue and help to guide people for comfortable viewing.