Abstract: Objective To get DNA aptamers binding to different subtype of breast cancer tissue and cells. Methods A random double-stranded DNA (dsDNA) pool was established by PCR, and single-stranded DNA (ssDNA) library was separated from dsDNA pool by coated Sepharose. Based on subtractive cell-SELEX, in vitro cultured breast cancer cells, MCF-7 and MDA-MB-231, were alternately used as positive screening target while normal mammary gland cell MCF-10A as negative. The effect of PCR amplification and the recovery of aptamer were confirmed by agarose electrophoresis. The PCR conditions were optimized through experiments of temperature gradient while the affinity of aptamers for target cells was monitored by using flow cytometry (FCM). Cloning and sequencing of aptamers were carried out by conventional molecular biology techniques. The aptamers were preliminarily screened by sequence analysis, and DNA aptamers were selected by flow cytometry, laser confocal and immunofluorescence staining. Results A random ssDNA library was successfully established and the screening conditions of SELEX were optimized. Specific DNA aptamers for MCF-7 and MDA-MB-231 cells were got after 20 runs’ tandem crossed cell-SELEX. Of 100 positive sequenced clones, 72 aptamers were identified. Accurate alignment of Blastn implied that not any similar sequence to 72 submitted aptamers was found in NCBI data bank. Five aptamers were preliminarily screened by sequence analysis, and two aptamers which could bind to breast cancer subtype tissue cells were obtained by optimization experiment. Conclusion By using tandem crossed cell-SELEX introduced in this article, DNA aptamers of broad-spectrum combing with clinical breast cancer subtypes can be obtained in a short time, and it is possible to explore the targeted drug delivery system of breast cancer.