This study presents a disposable, novel, and sensitive biosensing system to determine adiponectin, an obesity biomarker, in real human serum. The graphite paper (GP) working electrode is a new material for impedimetric biosensors. In the literature, there is no study in which this electrode is used in impedance-based biosensors for adiponectin detection. Sensitive and useful techniques, such as electrochemical impedance spectroscopy and cyclic voltammetry, were utilized for investigation of the modification of the GP electrode surface and optimization and characterization of the constructed biosensor. The single frequency impedance technique was used to study the interactions between antiadiponectin and adiponectin. The morphology of the electrode surface for each immobilization step was examined with scanning electron microscopy. All experimental parameters were optimized to fabricate a rapid and sensitive biosensing system. The designed biosensor presents excellent performance with a wide detection range (0.05-25 pg mL(-1)) and a low limit of detection (0.0033 pg mL(-1)) for adiponectin determination. Also, it has been demonstrated that the biosensor sensitively allows for the detection of adiponectin in human serum. The affinity of the designed immunosensor toward other proteins and components was examined in the presence of the target protein (adiponectin), leptin (100 pg mL(-1) creatine kinase (50 pg mL(-1)), parathyroid hormone (50 pg mL(-1)), and D-glucose (0.5 M). The selectivity of the adiponectin biosensor resulted in high capacity to neglect the interference effect. The constructed biosensor showed good linearity, long-term storage life (10 weeks), high reusability (18 times regenerability), and high ability to detect adiponectin concentrations at picogram levels.