The successful surgery of chronic otitis media (COM) is challenging; this depends on the surgeon’s knowledge of the optical visibility of surgical microscopes. Herein, we reported the utilization of intra-surgical optical coherence tomography (OCT) system to effectively guide the surgery of COM based on augmented reality with cross-sectional images. The intra-surgical spectral-domain OCT system with a center wavelength of 846 nm was capable of obtaining non-invasive, high-resolution, and high-speed visualizations with an axial resolution of 8 µm, lateral resolution of 30 µm, and an extended working distance of 280 mm. Three patients with COM were involved in this research. The lesion conditions of the temporal bone were observed with computed tomography pre-operatively. Furthermore, pure-tone audiogram examinations were performed to evaluate pre and post-surgical conditions. The results revealed that the averaged air-bone gap of 500 Hz, 1 kHz, 2 kHz, and 4 kHz in all cases improved to 61%. Thus, the research proves that the experimental procedure can be beneficial and clinically applicable with the developed intra-surgical OCT system for future otolaryngological assessments.