One of the possibilities of early detection of cancer disease is the analysis of the presence of protein biomarkers. Herein, we present the studies on the hybrid antibody—aptamer receptor layers for electrochemical detection of HER2 protein that is known as a biomarker of e.g. breast cancer. The application of MB-labelled aptamer did not allow to evidence the interaction with HER2 protein. Moreover, the use of unlabelled aptamer as a capture element and polyclonal antibody as a reporter probe did not show a dependence of current signal change vs HER2 protein concentration as well. On the contrary, the application of monoclonal antibody as a capture probe and aptamer labelled at 3′ end with MB as reporter probe enabled the determination of HER2 within 0.01–10 ng·ml−1 concentration range. The elaborated affinity assay also showed good selectivity towards HER2 biomarker and was used for HER2 determination in spiked serum sample.