A label-free microfluidic method for separation and enrichment of human breast cancer cells is presented using cell adhesion as a physical marker. To maximize the adhesion difference between human breast epithelial cells (MCF10A) and cancer cells (MCF7), flat or nanostructured polymer surfaces (400nm pillars, perpendicular, or parallel line) were constructed on the bottom of PDMS microfluidic channels using UVassisted capillary moulding. The adhesion of MCF10A and MCF7 on each channel was measured based on detachment assays where the adherent cells were counted with increasing flow rate after a pre-culture for a period of time (e.g., 1, 2, and 4 hrs). It was found that MCF10A cells show higher adhesion than MCF7 cells regardless of surface nanotopography. The optimum separation was found for 2 hours pre-culture on the 400nm perpendicular line pattern at a flow rate of 200 µl/min. The fraction of MCF7 cells was increased from 0.36 ± 0.04 to 0.83 ± 0.04 under these optimized conditions.