Tumour invasion into the surrounding stroma is a critical step in metastasis, and it is necessary to clarify the role of microenvironmental factors in tumour invasion. We present a microfluidic system that simulated and controlled multi-factors of the tumour microenvironment for three-dimensional (3D) assessment of tumour invasion into the stroma. The simultaneous, precise and continuous arrangement of two 3D matrices was visualised to observe the migration of cancer cell populations or single cells by transfecting cells with a fluorescent protein. A vascular endothelial layer was formed to simulate transendothelial transport of nutrients, and its endothelial barrier function was verified by the diffusion of 70 kDa fluorescein isothiocyanate (FITC)-Dextran in 3D matrices. Through high-throughput cell migration tracking observation and statistic evaluation, we clarified that cell density of the tumour directly determined its invasiveness. The results suggested that increased secretion of IL-6 among both cancer cells (MDA-MB-231) and noncancerous cells (MCF-10A or HDF-n) after co-culture contributes to cancer cell invasiveness, and this was verified by an IL-6 inhibitor assay. Finally, the drug efficacy of paclitaxel was reflected as changes in cancer cell migration ability, viability, and morphology. Together, our microfluidic devices could be a useful tool to study the mechanism of tumour invasion into the stroma and to screen anti-metastatic drugs.