Near-infrared surface-plasmon resonance (SPR) spectroscopy can be used to determine the thickness of single organic molecular films and the permittivities of organic materials with a high degree of sensitivity. The two-wavelength SPR method with one NIR and one visible light probes was used to improve the accuracy of our experiment. Structures of organic molecules on various substrates were investigated. Particular attention was focused on the error in the measuring of thickness, which was caused by the surface roughness of the substrate. The effect of this was determined by ATM observations. The error in measurement caused by a rough substrate was, thus, estimated.