A toluidine blue O (TBO) terminated self-assembled monolayer (SAM-I) was fabricated on a gold disk electrode by nucleophilic attack of the amino group in the reduced TBO on an immobilized carbonyl moiety of terephthaloyl chloride with cyclic scanning. The TBO units in the SAM-I were observed by surface eahanced Raman spectroscopy (SERS). The average TBO surface coverage of SAM-I was (2.5 +/- 0.5) x 10(-10) mol cm(-2). Meanwhile, another TBO derived SAM (SAM-II) was prepared for comparison by activating the free carboxyl group of bound terephthaloyl chloride and then reacting it with the amino group of TBO. The average TBO surface coverage of SAM-II was only (6.8 +/- 0.1) x 10(-11) mol cm(-2). Both SAMs possessed good electroactivity and showed identical properties. With an electrooxidative coupling of TBO units in SAM-I, a novel electroactive polymerized TBO monolayer (Film-I) was constructed, which displayed an improved stability and obvious electrochemical blocking effect. As a comparison, a poly(TBO) film (Film-II) was obtained by the electrochemical polymerization of TBO in solution on SAM-I for remedying the defectiveness of the knitted structure of Film-I. The SERS displayed more TBO units in Film-II than in Film-I. Film-II provided a much better barrier to the redox probe of K-3[Fe(CN)(6)] due to the formation of the perfect structure.