Leptin is a peptide hormone produced primarily in adipose tissues. Leptin is considered a biomarker associated with obesity and obesity-mediated diseases. Biosensor detection of leptin in the blood may play a critical role as an indicator of dynamic pathological changes. In this paper, we introduce an electrochemical biosensor that adopts o-Phenylenediamine (oPD) on screen-printed gold electrodes (SPGEs) for detecting the leptin from a mouse model of diet-induced obesity (DIO). A linear calibration curve for the leptin concentration was obtained in the ranges from 0.1 to 20 ng/mL with a lower detection limit of 0.033 ng/mL. The leptin concentration was quantified with HRP (horseradish peroxidase)-catalyzed oxidation of oPD by two voltammetry methods: cyclic voltammetry (CV) and square-wave voltammetry (SWV). The proposed sandwich enzyme-linked immunosorbent assay (ELISA)-based electrochemical biosensor for the leptin in mouse blood serum showed high stability, sensitivity, selectivity, and effectivity compared to the commercial Leptin ELISA measurement. Thus, we believe that this leptin biosensor can be a sensitive analytical tool to detect low-levels of biomarkers in clinics and point-of-care testing (POCT).