Extracellular matrix (ECM) stiffness plays a crucial role in cell mechanobiology. Culturing cells in 3D better recapitulates the in vivo conditions. However, there are few platforms allowing measurements of the dynamics of 3D ECM stiffness resulting from cell remodeling activities. Ultrasound shear wave elasticity imaging (SWEI) is a promising tool to evaluate the spatiotemporal change of ECM stiffness. The aim of this study is to evaluate the feasibility of quantifying changes of ECM stiffness in a 3D cell culturing system using SWEI. Collagen-based 3D cell culture complexes suitable for SWEI were developed. The cell-culturing complex was mounted to a SWEI system consisting of a 20 MHz push transducer for shear wave (SW) generation and a 40 MHz imaging transducer motorized by a mechanical scanner. The change of complex thickness resulting from cell remodeling was determined by B-mode images. A spatiotemporal map of matrix displacement resulting from SW propagation was generated and converted into a k-space map by 2D discreet fast Fourier transform. The dispersion curve, defined as the function of SW speeds with respect to frequencies, was fitted by viscoelastic models to estimate the shear moduli. Using this novel SWEI platform, we successfully demonstrated changes of matrix stiffness and structure when culturing the system with different cancer and normal cell lines. The cultured cells actively remodeled the ECM with significant contraction of matrix, with that a higher degree of matrix contraction corresponded to a larger shear modulus. Biochemical tests revealed the matrix stiffening mediated by human adenocarcinoma cells CL1-5 mainly resulted from cell contraction and cross-linking of the matrix proteins. Our results indicate the potential of our system in identifying the mechanistic details of various pathological conditions that are associated with change of matrix stiffness.