We developed a method to measure the thickness profile and refractive index variation for silicon wafers based on a spectral-domain interferometry. Through a spectral domain analysis of multiple interferograms obtained using a femtosecond pulse laser, two physical quantities, the thickness profile and the refractive index variation can be measured at high speed. For practical realization, two factors affecting motion errors were considered, long-term drift of the optical path difference and measurement repeatability with dynamic motion. The proposed system was demonstrated with two translation stages and the thickness profile and refractive index variation of a 100 mm silicon wafer along its center line were measured. The measured thickness profile showed a wedge-like shape with a maximum deviation of 2.03 μm at an average geometrical thickness of 478.03 μm. The mean value and the deviation of the refractive index distribution were 3.603 and 0.005, respectively. To evaluate the performance of the proposed system, the measurement repeatability and the measurement uncertainties were estimated.