Shear wave elastography (SWE) has been widely adopted for clinical in vivo imaging of tissue elasticity for disease diagnosis, and this modality can be a valuable tool for in vitro mechanobiology studies but its full potential has yet to be explored. Here we present a laser speckle contrast SWE system for noncontact monitoring the spatiotemporal changes of the extracellular matrix (ECM) stiffness in three-dimensional cancer cell culture system while providing submillimeter spatial resolution and temporal resolution of 10 s. The shear modulus measured was found to be strongly correlated with the ECM fiber density in two types of cell culture system (r = 0.832 with P < 0.001, and r = 0.642 with P = 0.024 for cell culture systems containing 4 mg/ml Matrigel with 1 mg/ml and 2 mg/ml collagen type I hydrogel, respectively). Cell migration along the stiffness gradient in the cell culture system and an association between cell proliferation and the local ECM stiffness was observed. As the elasticity measurement is performed without the need of exogenous probes, the proposed method can be used to study how the microenvironmental stiffness interacts with cancer cell behaviors without possible adverse effects of the exogenous particles, and could potentially be an effective screening tool when developing new treatment strategies.