Based on spectral domain optical coherence tomography with ultra-wideband light source, one Doppler shift calibration method suitable for a practical system is proposed, in which the mirror motion in the traditional method is replaced by a moving optical-path module, thereby enabling its applications in actual situations. After acquiring a series of time-varying spectra in motion, the proposed method is used to analyze the spectral distribution range via Fourier transform. Compared with the traditional calibration method, the proposed method is more precise and the axial resolution of the system is improved by 0.34 μm. Thus, this method improves the imaging effect in actual biological tissue imaging.