In spectral-domain optical coherence tomography (SD-OCT), the limited resolution of the spectrometer causes nonuniformity of the interference signal. The latter, in turn, causes the sensitivity of SD-OCT to decrease, thereby limiting the imaging range and decreasing the axial resolution. We addressed this problem by applying nonuniform, nonharmonic analysis (NUNHA) with software that features high-frequency resolution without interpolation. We demonstrate the application of NUNHA in SD-OCT and compare it with conventional frequency analysis methods by simulating nonuniform interference signals. The results suggest that application of NUNHA in SD-OCT can provide acquisition of a clearer tomographic image, accurate analysis of fine and complex structures, and preservation of resolution and sensitivity at regions deep within a sample. This is because it reduces the influence of nonuniformity caused by the spectrometer and is unaffected by distortion due to interpolation.