The organothiol 4-mercaptopyridine (pyS) has been used extensively as facilitator for the assessment of heterogeneous electron transfer reaction of cytochrome c (cyt c). Its efficiency, however, is strongly affected by the instability of the adlayer due to the C-S bond cleavage. The K-4[Ru(CN)(5)(pyS)]. 3H(2)O complex was synthesized and characterized aiming its utilization as an inorganic self-assembled monolayer (SAM) that would enhance the gold adlayer stability. The SAM formed by this complex onto gold (RupySAu) was characterized by spectroscopic (FTIRRAS and SERS) and electrochemical (LSV) techniques. The ex situ vibrational SERS and FTIRRAS spectra data of this SAM formed onto gold suggest a sigma interaction between the gold and sulfur atoms of the complex, inducing a perpendicular arrangement in relation to the surface normal. Additionally, SERS and FTIRRAS spectra performed for freshly prepared RupySAu adlayer and for large immersion times in the precursor solution have not shown any significant change that would reflect the degradation of the adlayer. The LSV desorption curves of this SAM indicate an enhancement in the C-S bond strength of the pyS ligand when coordinated to the [Ru(CN)(5)](3-) moiety. Comparatively to the data obtained for the desorption process of the pyS monolayer, the reductive desorption potential, E-rd, of the RupySAu presents a shift of -17 mV. This bond strength intensification leads to an increase in the stability of the monolayer. The voltametric curves of cyt c carried out with the RupySAu electrode showed electrochemical parameters consistent with those reported for the native protein, as well as the maintenance of the electrochemical kinetic data after repetitive cycles. The results all together suggest that the pi back-bonding effect from the [Ru(CN)(5)](3-) metal center plays an important role in the stability of the RupySAu adlayer, improving the assessment of the cyt c heterogeneous electron transfer reaction.