Electrochemical label free DNA hybridization discrimination of the brain tumor sequence CK20 has been made at the gold-thiol and thiol diluent binary and ternary mixed monolayer interfaces in presence of the [Fe(CN)6](3-) and double stranded DNA (dsDNA) specific cationic intercalators, proflavine (PF) and methylene blue (MB), respectively. Thiol hexane labeled single stranded DNA (HS-ssDNA) and thiol diluents such as 6-mercapto-1-hexanol (MCH) and 3-mercaptopropionic acid (MPA) are used to construct the mixed monolayers. Change in the peak-to-peak separation (Delta Ep) for the [Fe(CN)6](3-) redox reaction indicates the efficiency of the diluents in removing the randomly oriented HS-ssDNA. Smaller Delta Ep 248 mV noticed for the HS-ssDNA/MPA compared to the HS-ssDNA/MCH mixed monolayers (812 mV) indicates the less influence of the MCH diluent on the arrangement of HS-ssDNA layer. However, the hybridization discrimination effect negotiated in presence of both the [Fe(CN)6](3-) and PF intercalator showed zero effect for the HS-ssDNA/MPA interface, and approximately 20-30% effect for the HS-ssDNA/MCH interface. The discrimination effect at the HS-ssDNA/MPA interface further increased to 80% by inserting the MCH at the local defects to form a multicomponent ternary HS-ssDNA/MPA/MCH layer interface. These differential discrimination effects are attributed to the formation of compact and/or defective layer structures, evidenced from their reductive desorption voltammetry in 0.5M KOH. The presence of single base (C-A) mismatch in the hybrid is diagnosed by a decrease in coulometric charge compared to the perfect dsDNA. The target concentration of 10 pM is detected selectively and sensitively.