An aptamer-based assay is described for the determination of trace levels of the cancer marker human epidermal growth factor receptor-2 (HER2) and living MCF-7 cells. The method is based on the use of a bimetallic MnFe Prussian blue analogue coupled to gold nanoparticles (MnFePBA@AuNP). Compared to pristine MnFe PBA nanocubes, the series of MnFePBA@AuNP exhibits a core-shell spherical nanostructure, and the shell thickness decreases from 99.9 nm down to 49.3 nm on increasing the fraction of AuNPs. The composite was placed on a gold electrode and incubated with the aptamer solution through electrostatic interaction. Then the modified electrode was employed to detect HER2 and MCF-7 cells using [Fe(CN)] as redox probe and displays good responses to both of them. Electrochemical impedance spectroscopy data show that the signal variation between each step during the whole procedure for the HER2 and MCF-7 cells detection can be embodied as the resistance value change between the [Fe(CN)] and electrode surface. The assay has a very low detection limit (0.247 pg∙mL) and works in the 0.001-1.0 ng∙mL HER2 concentration range. It was also used to sense HER2 in MCF-7 cells, and this results in an assay that works within the 500-5 × 10 cell∙mL cell concentration range and a 36 cell∙mL detection limit. Furthermore, the aptamer-based assay is selective, acceptably reproducible, stable, and well feasible for the detection of HER2 and living MCF-7 cells in human serum. Graphical abstract Schematic of an electrochemical aptasensor based on the bimetallic MnFe Prussian blue analogue (MnFe PBA) coupling with gold nanoparticles (represented by MnFePBA@AuNPs). It was employed as the aptasensor for human epidermal growth factor receptor-2 (HER2), and living MCF-7 cells.