Abstract A non-destructive method for measuring the physical thickness and group refractive index of individual layers was proposed based on spectral-domain interferometry, which was realized to achieve real-time measurements using a mode-locked laser and an optical spectrum analyzer. As a double-stacked specimen, a microfluidic channel mold composed of a SU-8 photoresist and a silicon wafer was chosen. With areal scanning of the sample, a physical thickness map and a representative group refractive index value for each layer were obtained at the same time. The sample was measured 30 times consecutively at pre-determined points to estimate the repeatability of the physical thickness, for which the standard deviation was less than 10 nm. Moreover, a measurement comparison with two calibrated reference instruments was conducted. According to the comparative measurement results, physical thickness values obtained by the proposed method and with each comparative method were found to be in good agreement within expanded uncertainty levels.