Cancer metastatic dissemination is a complex event during tumor progression which involves cell-cell and cell-matrix interactions. Micropatterning is one of the most efficient ways to study tumor development because it can tune the distribution of cells with spatial and temporal control. Extensive studies have shown that microfluidics can provide a feasible method for cell patterning. However, the current technique requires a microfabrication laboratory to manufacture the chip, which results in inaccessibility to researchers, especially biologists who focus on disclosing biological mechanisms rather than the methods. In this work, we developed a new methodology (tape-assisted photolithographic-free microfluidic chip, TAPMiC) that can realize homogeneous and heterogeneous micropatterning (45 features, 300 μm diameter of each) on a culture dish without the photolithographic procedure. We have applied this method to study critical biological problems, such as tumor cell migration under different conditions, including antitumor pharmaceutics and candidate gene RNAi assay that was relevant to tumor translocation and invasion. Moreover, this platform can achieve copatterning to recapitulate the tumor invasion scenario with single-cell trackable analysis. To decode regulation during metastasis, we conducted in situ recovering for quantitative polymerase chain reaction (qPCR) analysis from each cell type from tumor-fibroblast copairing. Regulation of several essential genes has unveiled that matrix degradation gene MMP2 and angiogenesis associated gene VEGFA were up-regulated in tumor cells in the fibroblast-enriched niche compared with homogeneous cultivation. Therefore, this approach constitutes a novel tool for investigating metastasis with quantitative measurements both on phenotype and genetical information.