Bioelectrochemical sensing of Mycobacterium tuberculosis through electro-immunosensors is a promising technique to detect relevant analytes. In general, immunosensors require the formation of organic assemblies by the adsorption of molecular constituents. Moreover, they depend on the correct immobilization of the bio-recognition element in the biosensor. These procedures cannot be easily monitored without the use of invasive methods. In this work, an impedance analysis technique was used, as a non-invasive method, to measure and differentiate the manufacturing stages of the sensors. Biomicrosystems were fabricated through physical vapor deposition (PVD) of 80 nm Au nanolayers on 35 µm copper surfaces. Later, the surface was modified through thiolation methods generating a self-assembled-monolayer (SAM) with 20 mM 4-aminothiophenol (4-ATP) on which a polyclonal antibody (pAb) was covalently attached. Using impedance analysis, every step of the electro-immunosensor fabrication protocol was characterized using 40 independent replicas. Results showed that, compared to the negative controls, distilled water, and 0.5 µg/mL HSA, a maximum variation of 171% between each replica was achieved when compared to samples containing 0.5 µg/mL of ESAT-6 M. tuberculosis immunodominant protein. Therefore, this development validates a non-invasive method to electrically monitor the assembly process of electro-immunosensors and a tool for its further measure for detection of relevant antigens.