Estrogens, a group of steroid compounds functioning as the primary female sex hormone, play an important role in the development and progression of breast cancer. In this study, using a novel annealing control primer-based GeneFishing PCR technology, five differentially expressed genes (DEGs), expressed using 10nM mitogenic estrogens, $17{eta}$-estradiol (E2) and $16{alpha}$-hydroxyestrone ($16{alpha}$-OHE1), were selected from the estrogen receptor (ER)-positive MCF-7 human breast cancer cells. The DEGs, MRPL42, TUBA1B, SSBP1, KNCT2, and RUVBL1, were identified by comparison with the known genes via direct sequencing and sequence homology search in BLAST. Quantitative real-time PCR data showed that two DEGs, tubulin ${alpha}1b$ and kinetochore associated 2, were greater than 2-fold upregulated by E2 or $16{alpha}$-OHE1. Both genes could be new biomarkers for the treatment and prognosis of cancers, and further study may provide insights into the molecular mechanisms underlying development and progression of breast cancer.