Breast cancer has multiple molecular subtypes; normal-like, basal-like, luminal A, luminal B and HER2 positive depending on receptor status of tumor cells. Cancer therapy is tailored according to the type of cancer; hence finding new diagnostic markers is important to decide on the best treatment approach. Cholinergic nicotinic receptor alpha 5 (CHRNA5) is one of the subunits of nicotinic acetylcholine receptors with significant roles in addiction and cancer. In the present study, CHRNA5 has been validated as an estrogen and/or Estrogen receptor (ER) modulated nicotinic acetylcholine receptor by qPCR in in vitro and in vivo in breast cancer samples. CHRNA5 isoform expression was measured using in vitro cell culture studies in which ER- and ER+ cell lines treated with different doses of estradiol (E2); MCF7 cell line was exposed to long-term E2 depletion, in another experiment it was treated with tamoxifen (4-OHT), an ER antagonist, and with or without E2. We found that all CHRNA5 isoforms exhibited increased expression in response to E2 dose-dependently in the ER+ MCF7 cell line while in the ER- MDAMB-231 cell line CHRNA5 isoform expression response was variable in direction and magnitude. CHRNA5 isoform expression in general steadily decreased in ER+ cell line MCF7 after 4-OHT treatment. After six months of E2 depletion, ER+ MCF7 cell line had increased CHRNA5_v3 isoform and ESR1 (ER gene) mRNA expression. In vivo, a human breast cancer cDNA panel was scanned with specially designed primers with qPCR using a custom-written GUI in MATLAB. It was found that CHRNA5, showing a statistically significant difference between normal and tumor cDNA, was a good candidate gene in diagnosis of breast cancer. CHRNA_v3 was able to distinguish between ER+ vs ER- breast tumor samples. We also addressed whether CHRNA5 isoforms exhibited differences in distinguishing tumor stage, and HER2 status. Our findings showed that expression of CHRNA5 isoforms were correlated with each other and regulated by E2 in breast cancer depending on ER receptor status.