To optimize the flicker phenomenon in active shutter type stereoscopic display, the influence of ambient light is deeply investigated. The transient luminance response measurement system with high sampling rate is used to measure and record ambient light through shutter glasses in real time. The measured time domain signal is transformed into frequency domain through Fourier transformation for the analysis of possible flicker source. Meanwhile, two perception experiments are implemented based on the multi-parameter adjustable light emitting diode (LED) lighting system. The experiment result shows that the involved factors such as frequency, maximum intensity, duty cycle and displayed image on the display, all have significant effect on the ambient light flicker felt by human eye through shutter glass, and 120 Hz or higher times frequency as matching ambient light can eliminate the flicker caused by ambient light and accomplish better visual comfort. The investigation result guides and facilitates the optimization of flicker in stereoscopic display with active shutter glasses and the improvement of visual comfort.