Gold electrodes are commonly used in electrochemical biosensors due to their welldescribed functionalization process with thiols and good chemical stability. However, the use of the commonly used redox-pair in electrochemical impedance spectroscopy, Hexacyanoferrate (II)/(III), can lead to corrosion of the gold electrodes, which damages the surface modification and alters the sensing signals. To address this issue, the authors introduced the in-situ generation of Hexaammineruthenium (II)/(III) as a redox-pair during the impedimetric measurement by applying a DC-bias. The DC-bias was chosen to supply Hexaammineruthenium (II) in a suitable concentration at the electrode surface by reducing Hexaammineruthenium (III). In this study, the stability of photolithographically fabricated thin-film and screen-printed gold electrodes was compared in Hexacyanoferrate and Hexaammineruthenium solutions. Long-time characterization of the electrochemical properties with cyclic voltammetry and electrochemical impedance spectroscopy revealed that Hexaammineruthenium (II)/(III) was an excellent redox-pair for stable impedimetric measurements with gold electrodes. To demonstrate the suitability of Hexaammineruthenium for biosensing, the authors applied it for the impedimetric detection of human-IgG. This biosensor exhibited a linear range from 11.3 ng/mL to 113 μg/mL, which is a suitable range for diagnostic applications. The use of in-situ generated Hexaammineruthenium (III) as an alternative redox-probe to replace HCF in electrochemical impedance spectroscopy for the detection of biomolecules using gold electrodes significantly improved the stability of the sensor and reduced the risk of false sensor signals. Furthermore, Hexaammineruthenium (III) was applied as a redox-probe to realize a human-IgG biosensor based on EIS, and the measured signals were stable, showing a good linear correlation to the logarithm of IgG-concentrations over five decades. This study has successfully introduced Hexaammineruthenium (III) as an alternative redox-probe to replace HCF in electrochemical impedance spectroscopy for the detection of biomolecules using gold electrodes. The use of a DC-bias during the EIS measurement generated Hexaammineruthenium (II)/(III) and did not cause a degradation of the gold electrodes, allowing recording of stable EIS signals for at least 60 minutes. The RCT value decreased only by 10%, in comparison to a variation between 100% and 140% when HCF was used as a redox-pair measuring a constant analyte concentration. Neither the production method nor the surface coverage of the sensor resulted in sufficiently stable gold electrodes for EIS measurements with HCF as a redox-probe. The authors believe that additional optimization of the functionalization and the DC-bias can improve the sensor performance even further. An important next step will be the application of Hexaammineruthenium for EIS biosensing in biological samples such as serum and saliva, which will further validate the effectiveness of this alternative redox-probe. Overall, this approach could lead to the employment of the EIS technique as a reliable biosensing method for point-of-care testing applications.