Objective：To get DNA aptamers binding to different subtypes of breast cancer cells. Methods：A random double - stranded DNA（dsDNA）pool was established by PCR，and single-stranded DNA（ssDNA）library was separated from the dsDNA pool by coated sepharose. Based on subtractive cell - systematic evolution of ligands by exponential enrichment（SELEX），the in vitro cultured breast cancer cells，MCF -7 and MDA -MB -231，were alternately used as positive screening targets 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 temperature gradient experiment，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 FCM，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 obtained after 20 runs’tandem crossed cell-SELEX. Of 100 positive sequenced clones，72 aptamers were identified. Accurate alignment of Blastn implied no similar sequence to the 72 submitted aptamers. 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 the tandem crossed cell -SELEX，DNA aptamers of broad -spectrum combing with clinical breast cancer subtypes can be obtained in a short time，which may be helpful for the targeted drug delivery system of breast cancer.