An attempt was made to develop a new sensitive biosensor for pralatrexate, as an anticancer drug, based on its interaction with the guanine of fish sperm DNA anchored on a screen-printed electrode (SPE) modified with polypyrrole (PP)/octahedral Pd-doped Co3O4 composite (Oh-Pd-doped Co3O4 C). Electrochemical techniques like differential pulse voltammetry verified the mechanism of such an interaction on the dsDNA/PP/Oh-Pd-doped Co3O4 C/SPE surface. A reduction in the peak current of guanine oxidation elucidated the interaction in acetate buffer with pH = 4.8. The optimization of response was performed for the interaction method according to potential, accumulation time, reproducibility and drug content. The linear dynamic range was estimated at 1.0 nM to 150.0 μM as well as a limit of detection as low as 0.61 nM for the DNA and pralatrexate concentrations. The practical potential of the proposed sensor was verified by determining pralatrexate in its pharmaceutical matrices.