In the study, an interlayer was observed in a nano-meter scale SiO2 overlayer on Si substrate by X-ray reflectivity(XRR) analysis and a new method is introduced for the XRR analysis of SiO2 ultra-thin films on Si substrate. The normalized reflectivity curves were analyzed by fitting with the calculated reflectivity curves which were also normalized with the same reference curves. The XRR analyses show that the variation of the positions of the thickness fringes in the measured reflectivity curve is caused by the interference effect from two oxide layers of different refractive indices and of different thicknesses with each other. The result indicates that there exists a SiO2 interlayer of a different refractive index between the SiO2 overlayer and the Si substrate. The analytical method used in the study determines the thickness of a ultra-thin SiO2 layer on Si with low uncertainty.