A calibration technique for the three-dimensional measurement system of gas-liquid two phase flow based on a virtual stereo vision was researched. Firstly, a single high-speed camera and two groups of mirrors were used to construct the virtual stereo vision system and to establish a perspective transformation model for the high-speed camera and a virtual stereo vision three-dimensional measurement model. Then, the camera and the virtual stereo vision sensor in the virtual stereo vision system were calibrated. Finally, the distribution of the bubble in the water was simulated by a target standard ball and its space distance was regard as the measurement standards. Furthermore, the impacts of different calibration methods on the accuracy of three-dimensional reconstruction were compared. Experimental results indicate that the accuracy of the three-dimensional reconstruction is the best when the calibration reference is placed in a water tank and the left and right virtual cameras and sensors are calibrated, respectively. The absolute error and the relative error of the measurement distance are better than 0.13 mm and 0.49%, respectively. It suggests that the impact of the light splitting path and tube wall refraction on the calibration accuracy should be taken full account of in the calibration of the gas-liquid two-phase flow three-dimensional measurement system based on the virtual stereo vision system.