In vivo mechanical characterization of soft biological tissues has broad applications ranging from disease diagnosis to tissue engineering. Shear wave elastography based on the bulk wave theory has been widely used to measure the mechanical properties of soft tissues. Given that most soft tissues basically have layered structures, the dispersive feature of elastic waves should be considered when the thickness of the interested layer is comparable to or smaller than the wavelength. Bearing this fundamental issue in mind, we propose an ultrasound-based guided wave elastography (GWE) method to characterize the mechanical properties of layered soft tissues. The dispersion relations of guided waves in layered structures were derived first, and its explicit expression was achieved. An inverse approach based on the dispersion relation to characterize the mechanical properties of layered soft tissues was then established. Both finite element analysis (FEA) and phantom experiments were carried out to validate the new method. In vivo experiments on forearm skin demonstrate the usefulness of the present method in characterizing layered soft tissues. Statement of significance: Layered soft tissues and artificial soft materials are ubiquitous in both nature and engineering. Imaging their in vivo/in situ mechanical properties finds important applications and remains a great challenge to date. Here, we propose an ultrasound-based guided wave elastography method to in vivo/in situ characterize the elastic properties of layered soft materials. We validate the method via finite element analysis and phantom experiments and further demonstrate its usefulness in practice by performing in vivo measurements on forearm skins. Given that the dispersive feature of elastic waves in layered soft media is considered in our method, it provides the opportunity to assess the intrinsic elastic properties of an individual layer in a non-destructive manner as shown in our experiments.