This study presents a novel, single-use electrochemical biosensor for the leptin biomarker, which may have potential use for early diagnosis of childhood obesity. The graphite paper working electrode was used for the first time in impedimetric biosensors. All immobilization procedure, investigation of the optimal parameters and characterization of biosensors were followed and evaluated using Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV). The Scanning Electron Microscope (SEM) was utilized to visualize the morphology of the electrode surface during the immobilization steps of the immunosensor. Moreover, the characterization of the interactions between anti-leptin and leptin was investigated by using Single Frequency Technique (SFI). The applicability of the designed biosensor for real serum samples was tested for clinical use. It was observed that the biosensor allows high sensitivity in the analyte detection (leptin) in real serum samples. Moreover, it was suggested that the developed biosensor presents advantages such as long shelf life (5% loss of activity after 8 weeks and 60% loss after 10 weeks), ability to determine analyte concentrations at picogram level (0.2 pg mL(-1) -20 pg mL(-1)), low limit of detection (0.00813 pg mL(-1)), reproducibility, reusability (12 times) and high sensitivity.