Detection of specific sequences of target DNA is of high importance in many fields, especially in medicinal diagnostics. This study focuses on the response of an electrochemical, label-free DNA sensor at two different hybridization temperatures (37 and 44 degrees C). The stem-loop structured probes and the blocking polyethylene glycol molecules were self-assembled on the electrode through S-Au bonding, to form a mixed monolayer employed as the sensing platform. Impedance spectroscopy was used for investigation of the electron transfer processes at a modified gold electrode before and after hybridization with the target DNA. The sensor showed sensitive and selective detection of the target DNA at the lower temperature, whereas the higher temperature affected the dynamics of the hairpin significantly, reflected in an increased sensitivity of the sensor.