We presented an integrated microfluidic system for dynamical study of cell-microenvironmental interactions. We demonstrated its precisely spatio-temporal control in the flow direction and the multi-site staying of the fluids by groups of monolithic microfabricated valves through digital operation, aside from the regulated communication between two loci based on real-time microenvironment transition. Using this system, a series of functional manipulations, including specific delivery, addressable surface treatment, positional cell loading and co-culture were performed quickly and efficiently for biological applications. Sequentially, we carried out the potential utility of this system in the research of dynamic microenvironmental influence to cells using a patho-physiological interaction during cancer initiation and progression. Our results exhibit the passive role but collaborative response of NIH 3T3 fibroblasts to the soluble signals from hepatocellular carcinoma cells, and also the variable behaviors of carcinoma cells under different environmental stimulation. This system can facilitate the in vitro investigation of cell-microenvironmental interactions occurred in numerous biological and pathogenic processes.