A polydimethylsiloxane two-layer scaffold platform was designed to provide a three-dimensional biomimetic microsystem that allows the detection of epithelial-to-mesenchymal transition without the use of specific biomarkers. As a proof of concept, a novel microsystem that consisted of two layers of 15 µm thick grating structures was developed. These layers had gratings with 40 µm wide ridges and 10 µm wide trenches, and they were stacked together to form a scaffold platform. To investigate the feasibility of using the engineered platforms for detecting changes in epithelial-to-mesenchymal transition, transforming growth factor beta-1 was added to an untransformed nasopharyngeal epithelial cell line. On flat polydimethylsiloxane surfaces, transforming growth factor beta-1 did not significantly affect nasopharyngeal epithelial size, migration speed, or directionality. However, the effect of transforming growth factor beta-1 treatment on migration speed of nasopharyngeal epithelial cells cultured on the two-layer scaffold platform was significantly different. Furthermore, while almost no untreated nasopharyngeal epithelial cells could squeeze into the 10 µm wide trenches, 21% of the transforming growth factor beta-1 treated nasopharyngeal epithelial cells exhibited traversing behaviors on the two-layer scaffold platforms. Moreover, fibronectin coating on the trenches and bottom layers of the scaffold platforms further enhanced the transforming growth factor beta-1-induced traversing of nasopharyngeal epithelial cells into the narrow trenches. These results demonstrate that the engineered two-layer scaffold microsystem can be used to monitor epithelial-to-mesenchymal transition induced changes in cell migration and invasiveness, paving the way of using these platforms in high throughput drug screening.