In this work, we performed ozone treatment of isotropic pitch fibers to optimize the duration and temperature during the stabilization process. The treatment worked effectively for long-term and high-temperature stabilization. The isotropic pitch fibers were ozone-treated at different ozone concentrations of 10, 20, and 30 mg/l. The surface properties of the isotropic pitch fibers were analyzed by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy, while their structural characteristics were examined using X-ray diffraction. The experimental results indicated that the surface oxygen functional groups (COOH, OH, and CO) and oxygen concentration of the pitch fibers increased after the ozone treatment. During the oxidative stabilization process, the cross-linking reactions of the oxygen functional groups formed the thermosetting pitch, as confirmed by the replacement of hydrogen with oxygen. Additionally, it was observed that the ozone treatment could improve the optimized oxidative stabilization procedure in carbon fiber manufacturing. Therefore, the ozone treatment played an important role in optimizing the oxidative stabilization procedure of the pitch fibers.