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In this work, an electrochemical label-free DNA biosensor was developed for determination of levodopa (LD). The biosensor was constructed using reduced graphene oxide decorated with Fe3O4 magnetic nanoparticles (rGO–Fe3O4) on a carbon paste electrode (CPE) and double-stranded deoxyribonucleic acid (DNA) (DNA/rGO–Fe3O4-CPE). The application was related to the molecular interaction between LD and DNA. Thus, the voltammetric behavior of LD at the surface of DNA/rGO–Fe3O4-CPE was studied using differential pulse voltammetry (DPV) where the oxidation peak current of LD was measured as an analytical signal. A considerable increase was observed in the oxidation signal of LD at the DNA-coated electrode compared to the DNA-free electrode, indicating the pre-concentration of LD due to the interaction with the surface-confined DNA layer. Scanning electron microscopy, energy dispersive X-ray and Fourier transform infrared spectroscopy confirmed the structure of the synthesized nanocomposites (electrode composition). Electrochemical studies revealed that modification of the electrode significantly increases the oxidation peak currents of LD. Under the optimum conditions, the calibration curve was linear in the range of 0.5–600 nM with a detection limit of 0.11 nM. The relative standard deviation for 200.0 nM was 4.07% (n = 5). The developed biosensor was successfully applied to the analysis of LD in human serum and urine sample. [ABSTRACT FROM AUTHOR]