Zip nucleic acids (ZNAs) are new generation nucleic acids conjugated with repeated cationic spermine units that decrease electrostatic repulsions with the target nucleic acid strands. The spermine units increase their affinity to its target by decreasing electrostatic repulsion between negatively charged target DNA and probe itself. Consequently, a more stable and stronger nucleic acid hybridization could be obtained in the presence of full match of ZNA-DNA. A new generation nucleic acid ZNA biosensor was introduced for the first time in the present study with its implementation to the voltammetric detection of a single nucleotide mutation related to Factor V Leiden mutation. Sensitive and selective electrochemical analysis of Factor V Leiden mutation was performed by using carbon nanofiber (CNF) modified screen printed electrodes (SPE) and multi-channel screen printed array of electrodes (MULTIx8 CNF-SPEs) resulting in a relatively shorter time contrast to the conventional methods. The selectivity of ZNA probe over to mutation-free DNA sequences was also investigated. Under the optimum conditions, single-use ZNA biosensor was also tested in the presence of synthetic PCR samples containing a single base mutation/without mutation. Moreover, the hybridization of ZNA probe with its target was examined in the PCR template medium and accordingly, the results were compared.