An extensive study on cooperative interaction of Triplex Forming Oligonucleotides (TFOs) with a double strand DNA, to form a triplex-DNA structure at electrode surface, is here reported. The cooperative effect on triplex structure formation was assumed by the higher binding enthalpy value, calculated for the interaction between the duplex DNA structure and the TFO1 and TFO2 probes (-67.3 KJ/mol), respect the sum of the single duplex-TFO1 and duplex-TFO2 interactions (-47.0 kJ/mol). The formation of triplex-DNA structure was proven by kinetic modelling study performed using the Luzar and Chandler model. The results indicate that after 500 ns from interaction, H-bonds between the base pairs in the double strand DNA are weaken while new H-bonds between the TFOs and duplex DNA are formed. Molecular dynamic (MD) simulations indicate that the TFOs sequence distance (138 bps) and the amount of TA*T …