The aim of this work was to design and develop an electrochemical immunosensor based on electrospun carbon nanofiber (ECNF) mat for the detection of human epidermal growth factor receptor 2 (Her-2). First, ECNF mat was fabricated by electrospinning method and heat-treatment and used directly as a novel electrode. To determine Her-2, this ECNF mat electrode was modified by Au nanparticles (NPs), cysteamine (Cys) molecules, carbon nanotubes (CNTs), and antibody (Ab) molecules, respectively. Field emission scanning electron microscopy (FESEM) and energy-dispersive X-ray spectroscopy (EDS) analysis were used to characterize Au NPs modified ECNF mat electrode. In addition, analysis of Her-2 in spiked human blood serum samples were investigated by cyclic voltammetry (CV) using [Fe(CN)6]−3/−4 as a redox probe and the experiments were compared using commercial kit. The linear response range of the developed immunosensor was from 5 to 80 ng ml−1 with a limit of detection (LOD) of 0.45 ng ml−1. The results indicated that the designed immunosensor has high potential for the determination of Her-2 due to non-invasive, precise and rapid analysis. [ABSTRACT FROM AUTHOR]