Abstract. The present study aimed to identify polypeptides that specifically bond to breast cancer stem cells from a phage display random 12 peptide library, in addition to the affinity and specificity of polypeptides. A phage display random 12 peptide library was screened using breast cancer stem cells as targets isolated from the MDA-MB-231 cell line using the serum-free culture technique with hs578bst and MDA-MB-231 cells as subtract-screening cells. Positive and specific binding clones were amplified and sent for sequencing. The affinity and specificity of the positive clones were subsequently identified by ELISA and 3,3'-diaminobenzidine staining. The results demonstrated that phages were gathered ~500 times following three rounds of biopanning. ELISA identified that the affinity to breast cancer stem cells of the no. 6 phage was 6.14 times higher than that in the control group. In addition, immunohistochemistry observed that the no. 6 phage exhibited high-specificity bonding to breast cancer stem cells, and the peptide sequence of the positive phage was GYSASRSTIPGK following DNA sequencing and translation. Thus, the present study isolated a specific peptide that bonds to breast cancer stem cells from a phage display random peptide library, which may facilitate further studies regarding the stem cell-targeted therapy of breast cancer.