In this study, a DNA biosensor was constructed and used for the determination of phenazopyridine and D-penicillamine. This biosensor made of pretreated pencil graphite electrode (PGE) modi?ed with multiwall carbon nanotubes (MWCNTs) and chitosan (CHIT) decorated with ds-DNA. The interaction of phenazopyridine hydrochloride with ds-DNA based on the decreasing of the oxidation signal of guanine and adenine was studied electrochemically at a pencil graphite electrode (PGE) using differential pulse voltammetry. The difference between adenine and guanine signals of the ds-DNA after and before interaction with phenazopyridine hydrochloride was directly proportional to phenazopyridine hydrochloride concentration, which used for quantitative iections. Under the optimum conditions, a linear dependence of the guanine and adenine oxidation signals was observed for the phenazopyridine hydrochloride concentration in the range of 0.01–50 µg mL -1 with a detection limit of 0.003 µgmL -1 . This method is successfully applied for the determination of phenazopyridine hydrochloride in real samples. The second part, assessment of DNA damage caused by D-penicillamine in the presence of iron( III ). using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). Under the optimum conditions, a linear dependence of the guanine and adenine oxidation signals was observed for the phenazopyridine hydrochloride concentration in the range of 0.05–150 µg mL -1 with a detection limit of 0.005 µgmL ?1 .