The interaction of Efavirenz (EFV) with fish sperm dsDNA immobilized onto pencil graphite electrode (PGE) has been studied by using differential pulse voltammetric technique using an electrochemical DNA biosensor. The guanine signal was lower with (double stranded-DNA) dsDNA-treated PGE than the untreated electrode after the interaction with EFV occurred. The changes in the experimental parameters such as the accumulation time and the concentration of EFV were also studied. All necessary parameters such as sensitivity, selectivity, accuracy and precision were calculated. In addition, the detection and determination limits, reproducibility and applicability of the analysis to pharmaceutical dosage forms were also investigated. These results showed that this DNA biosensor could be used for the sensitive, rapid simple and cost effective detection and determination of EFV-dsDNA interaction. The linearity was between 2 and 24 ppm of EFV concentration on guanine signal decreasing curve. EFV showed an irreversible oxidation behavior at all investigated pH values. This oxidation step was adsorption controlled on PGE. Hence, differential pulse adsorptive stripping (AdsDPV) voltammetric method was developed for the determination of EFV. Accumulation time and potential were optimized. Under these conditions, the current showed a linear dependence with concentration in the range between 0.018 and 2.56 ppm. Both determination methods were fully validated and applied for the analysis of EFV pharmaceutical dosage form.