The response surface methodology (RSM) is used for optimization of foodborne pathogen detection based on label free electrochemical nucleic acid biosensors. Listeria monocytogenes amplicons obtained from food samples are used as model case. The extent of hybridization is determined by using guanine oxidation signals obtained with Differential Pulse Voltammetry and Electrochemical Impedance Spectroscopy. RSM is used to investigate the effects of hybridization parameters including target and salt concentration and hybridization time on the biosensor selectivity. The ratio of electrochemical transductions after hybridization with complementary and noncomplementary targets is the response of the statistical analysis obtaining a detection limit of 267 pM.