In this work, a cationic porphyrin, ascribed as [Ttolyl(P-(C6H5)3)4]4+, where Ttolyl = 5,10,15,20 tetrakistolylporphyrin, was electrostatically assembled with CdTe quantum dots capped with glutathione (GSH) of diameter ~3.28 nm via a layer-by-layer methodology. This multilayer assembly was evaluated as a biocompatible interface with synergic effects for calf thymus double-stranded DNA (CT DNA) immobilization, considering that the hybrid assembly contains a cationic porphyrin with a high binding constant for CT DNA, and quantum dots with polypeptide as a capping agent. The multilayer assembly, ascribed as ITO/{[Ttolyl(P-(C6H5)3)4]4+/CdTe} n (n = 1?5), was characterized by UV-Vis spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS). The last technique was explored in order to check the adsorption of CT DNA onto the multilayer porphyrin/quantum dot assembly. The difference in electron transfer resistance (?R ct) obtained after CT DNA incubations showed the best result for a specific multilayer assembly, n = 3. {[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 showed a good correlation of ?R ct with the logarithmic concentration of CT DNA, in the range 1.0 ? 10?10 to 1.0 ? 10?6 M with a limit of detection (LOD) of 1.5 ? 10?12 M. Conversely, when the {[Ttolyl(P-(C6H5)3)4]4+/CdTe}3 system was incubated with calf thymus single-stranded DNA and salmon testes DNA, no significant difference in ?R ct was observed. We conclude that the newly described {[Ttolyl(P-(C6H5)3)4]4+/CdTe} n system is a choice method for the impedimetric determination of CT DNA.