We present a simple, biomarker-free microfluidic device for separating cancer cells from a mixed solution of normal and cancer cells by difference in adhesion force. A polydimethylsiloxane (PDMS) microfluidic chip was fabricated onto glass substrate using standard soft lithography. Three types of polyurethane acrylate (PUA) nanostructure (50nm pillar, 50nm perpendicular groove, 50nm horizontal groove with respect to the direction of flow) were included inside the microfluidic channel by UV-assisted capillary molding. For cell types, MCF7 (breast cancer cell line) and MCF10A (breast normal cell line) were used. To find the optimum condition for separation, each cell line was injected into the microfluidic device and cultured for 1h, 2h, and 3h, respectively, followed by cell detachment by flow of medium solution with increasing flow rate. The adhesion force of MCF10A was stronger than that of MCF7. MCF10A cells cultured onto the nanopatterned surface were more spread than those cultured onto the glass surface. Furthermore, the presence of nanopatterns increased the ratio of adhesion force of normal and cancer cells and thus and the separation efficiency. The optimum culture condition was 2h onto the nanopattern and flow rate was ∼ 300μl/min.