A spectral calibration method for optical coherence tomography (OCT) is proposed. The method is implemented by introducing an interference signal with a fixed optical path difference (OPD) in the Fourier domain OCT system. The sample interference signal and the fixed OPD signal are acquired simultaneously. Then the fixed OPD signal is obtained from the mixed signals through a filter step, and its phases are calculated to calibrate the interference signal of the sample in the system. We theoretically analyze the feasibility of the proposed method. In a 100 kHz swept source OCT system, the spectral calibration experiments with a mirror as the sample are performed at different depth positions. The point spread functions and the axial resolutions are obtained. Compared with the results using the light source clock, the proposed method has a better effectiveness.