Nanoscaled thin films are typically deposited on various substrates to achieve their unique characteristics. These thin film systems can be affected by the thickness variations between the thin film and the accompanying substrate. To investigate the thickness dependence of a nanoscaled thin film system, ultrasonic atomic force microscopy (Ultrasonic-AFM) which can evaluate the localized elastic modulus using the contact resonance frequency of a vibrating cantilever is applied. Copper thin films of low elastic modulus were deposited on silicon substrates of high elastic modulus. By contrast, Si3N4 and Ti thin films of high elastic modulus were deposited on GaAs and glass substrates of low elastic modulus, respectively. Experimental results showed that the thin films with different thickness were affected significantly by the substrate and the contact resonance frequency changes as a result of the varying thickness of the thin films. This research demonstrate that Ultrasonic-AFM may be a novel technique for the nondestructive measurement of nanoscale thin film thickness by considering substrate features.