We introduce a theoretical framework for simultaneous refractive index and thickness measurement of multilayer systems using the Fourier domain optical coherence tomography (FD-OCT) system without any previous information about the item under investigation. The input data to the new formalism are the FD-OCT measured optical path lengths and properly selected spectral components of the FD-OCT interference spectrum. No additional arrangement, reference reflector, or mechanical scanning is needed in this approach. Simulation results show that the accuracy of the extracted parameters depends on the index contrast of the sample while it is insensitive to the sample’s thickness profile. For transparent biological samples with smooth interfaces, when the object is in an aqueous medium and has indices , this method can extract indices and thicknesses with the absolute error .