STM and impedance results of the self-assembled monolayer (SAM) formed with thionicotinamide (TNA) on gold indicate the presence of defects that increase with the immersion time of the electrode in the TNA solution affecting the SAM electroactivity toward the electron transfer reaction of the cytochrome c metalloprotein and [Fe(CN)6]4− and [Ru(NH3)6]3+ complexes. It was observed that this electroactivity was also affected by the pH of the electrolyte solution. SERS and STM data indicate sulfur coordination to the surface with contribution of the NH2 group. From the dependence of the TNA surface coverage on the temperature and concentration in solution, thermodynamic parameters of adsorption were determined.