An electrochemical nanostructure voltammetric sensor based on pencil graphite electrode modified with polypyrrole, nitrogen doped reduced graphene and ds-DNA as a label free DNA biosensor for determination of epirubicin and confirmed with docking simulation investigation. Display omitted •A highly sensitive DNA biosensor for nanomolar determination of epirubicin.•Synergistic effect of polypyrrole and nitrogen doped reduced graphene.•Docking simulations confirmed the interaction between DNA and epirubicin. Epirubicin is an effective anticancer drug, but naturally causes several side effects. Hence the determination of this drug in biological samples, plays a key role in evaluating its effects and side effects. In this research, a novel label-free DNA-based biosensor was fabricated for the analysis of epirubicin in biological samples. The sensor was fabricated through modifying a pencil graphite electrode with polypyrrole, nitrogen doped reduced graphene (for improving the electrical conductivity) and salmon sperm ds-DNA (PP/NrG/ds-DNA/PGE) and the changes in the electrode signal were used for the determination of epirubicin. The PP/NrG/ds-DNA/PGE showed a high sensitivity for epirubicin in the concentration range of 0.004–55.0 μM and had a detection limit of 1.0 nM. For further evaluations, the interactions between ds-DNA bases and epirubicin were investigated through a theoretical docking study and the obtained data confirmed the intercalation of epirubicin in the minor grooves of ds-DNA with guanine bases. The PP/NrG/ds-DNA/PGE was used for the analysis of epirubicin in injection and urine samples.