In order to ensure the accuracy and robustness of fish behavior data acquisition, an interference cancellation algorithm was proposed to optimize the 3D positioning algorithm of fish target.In this paper, the study was based on a singular phenomen-on caused by total reflection in this platform, the causes of the phenomenon were analyzed, and the influence of the phenomenon was eliminated. Firstly, based on the principle of geometrical optics and the principle of total reflection, the causes of interference phenomena were analyzed, and the characteristics of interference image were summarized to determine the elimination scheme.Using the digital image and video processing technology, the interference recognition and elimination algorithm was designed to optimize the original positioning algorithm.Based on the experiment of red crucian carp and simulated fish, the results showed that the proposed algorithm is accurate and confirmed that this algorithm has high precision and robustness.