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An electrochemical magnetic immunosensing strategy was developed for the determination of HER2-ECD, a breast cancer biomarker, and breast cancer cells in human serum. A sandwich assay was performed on carboxylic acid-functionalized magnetic beads (MBs) using a screen-printed carbon electrode (SPCE) as transducer surface. The affinity process was detected using electroactive labels; core/shell streptavidin-modified CdSe@ZnS Quantum Dots (QDs). Cd2+ ions, released from the QDs, were determined by differential pulse anodic stripping voltammetry (DPASV). An assay time of 90 min, with an actual hands-on time of about 20 min, a linear range between 0.50–50 ng·mL−1 of HER2-ECD and a limit of detection of 0.29 ng·mL−1 were achieved. Analysis of live breast cancer cells was also performed using the optimized assay. Breast cancer cell lines SK-BR-3 (a HER2-positive cell line), MDA-MB-231 (a HER2-negative cell line) and MCF-7 (a cell line with low HER2 expression) were tested. The selectivity of the assay towards SK-BR-3 cells was confirmed. A concentration-dependent signal that was 12.5× higher than the signal obtained for the HER2-negative cells (MDA-MB-231) and a limit of detection of 2 cells·mL−1 was obtained. [ABSTRACT FROM AUTHOR]