Three-dimensional (3-D) extracellular matrices (ECM) allow complex biochemical and biophysical interactions between cells and matrices. Unlike 2-D systems, 3-D models provide a better representation of the micro and local environments in living tissues for facilitating the physiological study of cell migration. Here, we report a microfluidic device based on polydimethylsiloxane (PDMS) for monitoring 3-D cell migration across ECM-coated microgaps with real-time light microscopy. We tracked the migration of the invasive MDA-MB-231 (mammary carcinoma) cells and mapped out their migration paths. It enabled us to quantify the percentage of migrated cells as well as migration information of individual cells. This wide spectrum of data acquisition is vital for elucidating the migration capabilities of different type of cells and to understand the basic mechanism involved in cancer metastasis.