Electrochemical DNA sensors are highly demanded for fast and reliable determination of antitumor drugs and chemotherapy monitoring. In this work, an impedimetric DNA sensor has been developed on the base of a phenylamino derivative of phenothiazine (PhTz). A glassy carbon electrode was covered with electrodeposited product of PhTz oxidation obtained through multiple scans of the potential. The addition of thiacalix[4]arene derivatives bearing four terminal carboxylic groups in the substituents of the lower rim improved the conditions of electropolymerization and affected the performance of the electrochemical sensor depending on the configuration of the macrocyclic core and molar ratio with PhTz molecules in the reaction medium. Following that, the deposition of DNA by physical adsorption was confirmed by atomic force microscopy and electrochemical impedance spectroscopy. The redox properties of the surface layer obtained changed the electron transfer resistance in the presence of doxorubicin due to its intercalating DNA helix and influencing charge distribution on the electrode interface. This made it possible to determine 3 pM–1 nM doxorubicin in 20 min incubation (limit of detection 1.0 pM). The DNA sensor developed was tested on a bovine serum protein solution, Ringer–Locke’s solution mimicking plasma electrolytes and commercial medication (doxorubicin-LANS) and showed a satisfactory recovery rate of 90–105%. The sensor could find applications in pharmacy and medical diagnostics for the assessment of drugs able to specifically bind to DNA.