Reagents binding specifically to target molecules are essential tools for clinical diagnosis and targeted therapy. Screening of target cell-surface specific affinity reagents with bench-top methods has some drawbacks, including time-consuming, labor-intensive and requirement of large-scale instrument. Microfluidic platforms may overcome these drawbacks because they could automate and complete the screening process within a shorter period of time. Phage display is a promising technology in selection of cell-surface specific peptides. In recent years, a subpopulation of tumor cells named cancer stem cells is believed to be the tumorigenic cells and closely associated with metastasis. A specific peptide that can recognize and differentiate cancer stem cells from the rest of cancer cell population is therefore useful for early diagnosis and targeted therapy. In this study, selection of M13 phage displayed peptides that bind with colon cancer cells and colon cancer stem cells using an integrated microfluidic system was successfully demonstrated. Compared with the traditional methods, the total selection process was shortened to 36 hours while traditional method needs almost a month. More importantly, the screening process can be automated and performed on a single microfluidic chip. The developed technique may be promising for early diagnosis of cancer and target therapeutics.