A new electrochemical immunosensor is developed for the label-free simultaneous detection of mucin1 (MUC1), cancer antigen 15-3 (CA15-3), and human epidermal growth factor receptor 2 (HER2) early breast cancer biomarkers. The biosensor is simply designed using the deposition of three different systems of redox species-antibody-conjugated polyethylenimine coated-gold nanoparticles (PEI-AuNPs), for the first time. The screen-printed carbon electrode (SPCE) comprising a three-working electrode array is modified with the conjugated PEI-AuNPs. Multiplex sensing is performed by utilizing the distinguishable electrochemical responses of the redox-active species; anthraquinone-2-carboxylic acid (AQ), thionine chloride (TH), and AgNO3 (Ag+) on the PEI-AuNPs conjugates for the detection of MUC1, CA15-3, and HER2, respectively. The single-run determination of the biomarkers by the proposed immunosensor is carried out by measuring the decrease (%) in the oxidation peak currents due to the formation of three kinds of antibody-antigen complexes. The decreased currents are logarithmically proportional to the antigen concentrations in the ranges of 0.10-100 U mL-1 CA15-3 and 0.10-100 ng mL-1 MUC1 and HER2 with detection limits of 0.21 U mL-1, 0.53 ng mL-1 and 0.50 ng mL-1, respectively, which are significantly lower than the clinically relevant cut-off levels. The sensor reveals high selectivity and satisfactory reproducibility. After storing for two weeks, the sensor retains the responses with ca. 90% of the initial currents. The immunosensor is successfully applied to detect three tumor markers in human serum and can provide a new technological platform for the development of low-cost, highly stable, sensitive, selective, and point-of-care (POC) diagnosis.