The combined measurement methods of subjective questionnaires, ECG signals, eye movement signals and ophthalmological parameters are used to study the effect of different types of display modes on visual fatigue of 2D and 3D TVs. The results show that the relative changes of BCDVA, TBUT, pupil diameter and Shannon entropy after watching 3D mode are significantly greater than those after watching 2D mode. Differences in relative RR interval values and heart rate (HR) induced by viewing 2D and 3D modes gradually increase after 50 min. Different types of display modes have different effects on subjective scoring symptoms, ophthalmological parameters, ECG parameters and eye movement parameters. The visual fatigue caused by watching 3D mode is greater than that caused by watching 2D mode, especially after watching 2D/3D mode 50 min, which has reference value for 3D movie production.