The authors describe a method for electrochemical determination of the breast cancer biomarker α-lactalbumin (α-LA) using disposable screen-printed carbon electrodes (SPCEs). Lysozyme-conjugated Fe3O4 nanoparticles (Lys-Fe3O4NPs) were used to capture α-LA on the surface of the SPCEs which then is trapped in an immunosandwich using secondary antibodies labeled with ferrocene-modified gold nanoparticles. The amperometric response of ferrocene (recorded at +0.1 V vs. silver pseudo-reference electrode) as well as the electrocatalytic activity of gold nanoparticles on the hydrogen evolution reaction (recorded at -1.0 V Vs Ag pseudo-reference electrode) was exploited to sense α-LA. A sensitive voltammetric response is observed, with (a) a sensitivity of 0.8789 μA·nM-1.cm-2, (b) a detection limit (LOD, at S/N = 3) as low as 0.07 ng·mL-1, and (c) linear response in the 0.75 to 630 ng mL-1 α-LA concentration range. The assay is selective and reproducible, and the SPCEs have good storage stability. The SPCEs were applied (a) to the analysis of (spiked) maternal milk, (b) of spiked serum from healthy and pregnant persons, and (c) of serum of patients suffering from breast cancer. Graphical abstract Schematic presentation of a sensitive electrochemical immunoassay platform based on ferrocene modified gold nanoparticles and lysozyme modified magnetic beads for the determination of alpha lactalbumin in human sera and breast milk by the amperometric response of ferrocene and hydrogen evolution reaction.