Abstract As carrier of genetic information, DNA is one of the most important intracellular targets that undergo modification and damage upon interaction with endogenous and exogenous factors. DNA is an excellent biomaterial for the construction of new devices, in nanotechnology and biosensor technology, for evaluation of DNA interaction with a broad range of chemical compounds and biomolecules, essential from a biological and a medical point of view. This review discusses recent advances on the design and applications of DNA-electrochemical biosensors that use DNA direct electrochemistry as a detection platform. AFM and voltammetric characterization of new bottom up immobilisation procedures of self-assembled nanostructures based on DNA single- and double-stranded, G-quadruplex, and i-motif configurations are presented, relevant for the development of new DNA-electrochemical biosensor devices. The applications of DNA-electrochemical biosensors, for the label-free detection of interactions with proteins, pharmaceutical compounds, metal ions and metal complexes, pollutants, free radicals, and electromagnetic radiation, were revisited.