The study on animal models to investigate different human diseases has made a remarkable contribution to the field of biomedical science. Many studies demonstrating the behavior activities of Drosophila have been studied with a motion tracking system. Most of these studies have been performed by the conventional paradigm where the Drosophila is held fixed by tethering or within the limited space. In this paper, we demonstrate the Active Omnidirectional Treadmill System (AOT) which enables the physically unrestricted Drosophila to navigate infinitely in two-dimensional space with the privilege of real-time tracking. The system maintains the position of the fly at a specific position by compensating the motion of the fly by counter direction motion of the sphere. The system is capable of maintaining the position error < 1000 µm for 89.7% of the time and the angular error < 5o 80.0% of the time. The study evaluates the performance of the different feedback control system through the experimental and simulation results. The successful phototaxis experiment was conducted to validate the usability of the system. The integration of the treadmill system with other peripherals for olfactory, vision and thermal stimuli can be a powerful tool to study the longitudinal behavior of the Drosophila. Further, the integration of the system with the optical microscopic device can be used to perform the brain imaging of the walking Drosophila.