Animal localization and trajectory tracking are of great value for the study of brain spatial cognition and navigation neural mechanisms. However, traditional optical lens video positioning techniques are limited in their scope due to factors such as camera perspective. For pigeons with excellent spatial cognition and navigation abilities, based on the beacon positioning technology, a three-dimensional (3D) trajectory positioning and tracking method suitable for large indoor spaces was proposed, and the corresponding positioning principle and hardware structure were provided. The results of in vitro and in vivo experiments showed that the system could achieve centimeter-level positioning and trajectory tracking of pigeons in a space of 360 cm × 200 cm × 245 cm. Compared with traditional optical lens video positioning techniques, this system has the advantages of large space, high precision, and high response speed. It not only helps to study the neural mechanisms of pigeon 3D spatial cognition and navigation, but also has high reference value for trajectory tracking of other animals.