A novel kind of ethylene-ferrocenyl dodecyl thiol bearing an electron-withdrawing pyridium group (FcCH=CH-Py(CH2)(10)SH) was synthesized and mixed with HOOCC10SH on an Au electrode to form mixed self-assembled monolayers (SAMs) in ethanol solution. The influence of the water content of acetonitrile on the electrochemistry of the SAMs was studied by cyclic voltammetry and ac voltammetry measurement. The results showed that the current decreased and the oxidation potential of the ferrocene group shifted negatively with increasing water content of acetonitrile.