A novel, selective, rapid and simple electrochemical method is developed for the determination of atropine sulfate. UV–Vis and differential pulse voltammetry are used to study the interaction of atropine sulfate with salmon sperm ds-DNA on the surface of salmon sperm ds-DNA modified-pencil graphite electrode (PGE). For this purpose, a pencil graphite electrode (PGE) modified with multiwall carbon nanotubes (MWCNTs), titanium dioxide nanoparticles (TiO2NPs), and poly-dialyldimethylammonium chloride (PDDA) decorated with ds-DNA is tested for the determination of atropine sulfate. The electrochemical oxidation peak current of adenine and guanine bonded on the surface of ds-DNA/PDDA–TiO2NPs–MWCNTs/PGE is used to obtain the analytical signal. Decreases in the intensities of guanine and adenine oxidation signals after their interaction with atropine sulfate are used as indicator signals for the sensitive determination of atropine sulfate. Using ds-DNA/PDDA–TiO2NPs–MWCNTs/PGE and based on the guanine signal, linear calibration curves were obtained in the range of 0.6 to 30.0μmolL−1 and 30.0 to 600.0μmolL−1 atropine sulfate with low detection limits of 30.0nmolL−1. The biosensor shows a good selectivity for the determination of atropine sulfate. Finally, the applicability of the biosensor is evaluated by measuring atropine sulfate in real samples with good accuracy.