A novel spectral calibration method is developed for spectral domain optical coherence tomography system. The method is based on two measurements of interference spectra from two reference mirror positions. It removes the influence of dispersion mismatch, and hence accurately determines the spectral distribution on the line-scan charge-coupled device (CCD) for sequent precise interpolation. High quality imaging can be realized with this method. Elimination of the degradation effect caused by dispersion mismatch is verified experimentally, and improved two-dimensional (2D) imaging of fresh orange pulp based on the proposed spectral calibration method is demonstrated.