Genetically encoded calcium indicators enabled to monitor activity of neurons with noninvasive manipulation; the technique has been shedding light on the unknown relationship between specific neural activity and its function. Combination of calcium imaging and automated tracking system further enabled to monitor activity of neurons even in freely moving worms, thus making it possible to dissect behavioral regulation by neural circuits. However, when we focus on interneurons, some methodological problems confront us. Since most of the interneuronal activity is appeared in a small neurite region, high-magnification observation is needed to monitor interneuronal activity. Then a very quick stage control is needed to keep a target region in a high-magnification microscopic field of freely moving animals. Moreover, focus change during the tracking in high-magnification is obstacle for acquiring enough quality of fluorescence images for estimation of calcium concentration change. To solve such problems, we have developed a high-speed tracking system that enables to track freely moving worms with high-magnification (50x) objective lens. Our system tracks an animal with high-speed (120fps) camera and continuous transparent light independent from fluorescence excitation light, thus photo-bleaching of fluorescence probes is reduced though keeping high-speed tracking. In addition, auto-focus system is implemented with three high-speed cameras with different focal depths that enable the system to perform high-speed autofocus by comparing simultaneously acquired images from the different depths. We demonstrate out system with the AIY interneurons that show prominent fluorescence intensity change in their neurites during the calcium imaging of fixed worms. AIY neurons are known to play important role in navigation, and therefore our system provides clues to understand function of interneuronal activity for behavioral regulation.