Although various sensors specifically developed for target analytes are available, affordable biosensing solutions with broad applicability are limited. In this study, a cost-effective biosensor for detecting human epidermal growth factor receptor 2 (HER2) was developed using custom-made gold leaf electrodes (GLEs). A novel strategy for antibody immobilization on a gold surface, mediated by protein L, was examined using commercial screen-printed gold electrodes and GLEs. This versatile immobilization strategy enables the application of the functionalized GLEs for the detection of antibodies and antigens. A self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) on the gold surface, was used to immobilize protein L, further binding trastuzumab to detect HER2 using electrochemical impedance spectroscopy (EIS). The HER2 detection was examined in phosphate-buffered saline (PBS) and supplemented cell culture medium. The modified GLEs showed good specificity and high sensitivity of HER2 detection without any enrichment steps, achieving a limit of detection (LOD) of 0.02 ng mL-1 in PBS and 0.03 ng mL-1 in cell culture medium, making the proposed immunosensor a cost-effective and sensitive solution for detection in complex biological matrices.