The aim of this work is preparation and use of an electrochemical DNA-based biosensor with a textured surface for the investigation of dsDNA host-guest interactions with specific chemicals in a biological sample of complex matrix such as urine. Results obtained at the search and optimization of polymer protective films over the DNA recognition layer at a glassy carbon electrode show that a polyvinyl alcohol membrane is the effective barrier able to stabilize the biosensor response in urine. The detection of acridines using the square-wave voltammetric (SWV) scan in the cathodic direction has allowed overcoming a problem of the electrochemical interference of DNA bases and acridines at the direct anodic scan. Height of the well-separated SWV peak at around 0.00 V to + 0.10 V (which corresponds to the reduction of the oxidation products of both of acridines) depends on the concentration of acridines. The peak potentials of acridines were shifted by about 0.10 V to the negative potential values in the urine medium compared to phosphate buffer. Intercalation of acridine yellow and proflavine into dsDNA was confirmed both in phosphate buffer and in urine.