Studies on the zebrafish behavior have attracted more and more attention. The algorithm of 3D coordinate calculation is the basis of zebrafish behavioral analysis. An automatic device for observing the behavior of zebrafish was designed based on binocular stereo vision technology. According to methods of p-rate threshold and pattern threshold, the image threshold algorithm was proposed. Horizontal X and Y coordinates of single zebrafish were figured out by calculating the average of pixel coordinates of image contour. If the difference of Y coordinate values among different zebrafishes is large, two groups of zebrafishes captured by two cameras will be set up separately according to the Y coordinate values, and the same serial number in two groups means the same target. If the difference of Y coordinate is small, the same target in different cameras was identified by the method of minimum distance. The formula of three-dimensional coordinates was conducted based on the refraction theorem and the structure of the observation device. Algorithm analysis shows that the running time of the algorithm proposed in the paper is saved. Validating testing to a number of fishes is designed and carried out to show that the calculated coordinates are close to the preset locations.