An electrochemical magnetoimmunosensor for the determination of estrogen receptor α (ERα) protein in complex samples (serum and cell lysates) able to discriminate between ERα positive and negative breast cancer cells is reported. Specifically functionalized magnetic microbeads with sandwich immunocomplexes and amperometric detection at disposable screen-printed carbon electrodes (SPCEs) resulted in highly selective and sensitive ERα detection with a detection limit of 19 pg mL−1. This magnetoimmunosensing platform was successfully applied to the quantitation of ERα in spiked human serum and cell lysates samples without any matrix effect with an advantageous performance in terms of simplicity and assay times over commercial ELISA assays. The biosensor capability for assessing ERα in intact breast cancer cells makes it competitive with conventional strategies providing rapidly quantitative and reliable results on this relevant biomarker currently used in the clinical practice for diagnosis, follow-up and monitoring of metastatic breast cancer. Keywords: Magnetoimmunosensor, Amperometry, ERα, Lysates, Intact cancer cells, Breast cancer