Estrogen receptors (ERs) are overexpressed in approximately 70% of breast cancer cases, and they play an important role in tumorigenesis. ERs are strong predictive factors for measuring responses to hormonal therapies. Aptamers are short and single stranded oligonucleotides that are able to recognize target molecules with high affinity. In the present study, we selected and synthesized an oligonucleotide, which has a similar sequence to estrogen response element in the Xenopus Vitellogenin A2 gene. The synthesized oligonucleotide was evaluated by using immunostaining of paraffin-embedded breast cancer tissues and treating MCF-7 human mammary carcinoma cell line in vitro. We found that the synthesized oligonucleotide had a high binding affinity to ER similar to estradiol. Using a specific anti-ER antibody as a standard control, we showed that the synthesized oligonucleotide specifically recognized and immunostained tumor cells of breast cancer without cross-reaction with normal tissues. The overall agreement of ER detection between the anti-ER antibody and the ER aptamer was 97.1% (kappa value=0.943; 95% CI=0.879-1.006; p<0.002). Similar to tamoxifen or fulvestrant, the oligonucleotide also had an inhibitory effect on cell proliferation of MCF-7 cell line in a dose- and time-dependent fashion but had no cytotoxic effect on human normal mammary epithelial cells. Therefore, the synthesized oligonucleotide may be used as an aptamer for immunostaining of paraffin-embedded tissue sections for breast cancer diagnosis, as well as a potential ER antagonist in the treatment of breast cancer.