Mosquitoes, as vectors of numerous serious infectious diseases, require rigorous behavior monitoring for effective disease prevention and control. Simultaneously, precise surveillance of flying insect behavior is also crucial in agricultural pest management. This study proposes a three-dimensional trajectory reconstruction method for mosquito behavior analysis based on video data. By employing multiple synchronized cameras to capture mosquito flight images, using background subtraction to extract moving targets, applying Kalman filtering to predict target states, and integrating the Hungarian algorithm for multi-target data association, the system can automatically reconstruct three-dimensional mosquito flight trajectories. Experimental results demonstrate that this approach achieves high-precision flight path reconstruction, with a detection accuracy exceeding 95%, an F1-score of 0.93, and fast processing speeds that enables real-time tracking. The mean error of three-dimensional trajectory reconstruction is only 10 ± 4 mm, offering significant improvements in detection accuracy, tracking robustness, and real-time performance over traditional two-dimensional methods. These findings provide technological support for optimizing vector control strategies and enhancing precision pest control and can be further extended to ecological monitoring and agricultural pest management, thus bearing substantial significance for both public health and agriculture.