Obesity diseases have drawn our attention attributing to unhealthy dietary habit. Leptin, as a peptide hormone produced primarily in adipose tissue, is a biomarker associated with obesity diseases. Here, we presented an electrochemical biosensor used reduced graphene oxide-gold nanomaterials (rGO-Au) to modify the glassy carbon electrode (GCE) and bionanocapsule-ZZ (ZZ-BNC) to vertically orient the antibody for detection of leptin. The linear correction curves of leptin concentration ranged from 0.001 to 1000 pg/mL with a low detection limit (0.00087 pg/mL). Compared with reported leptin sensors, the biosensor displayed low detection limit, higher selectivity and anti-interference. Notably, our biosensor enabled to detect the leptin in diet-induced obese mouse model with a good agreement of enzyme linked immunosorbent assay (ELISA). The distinct detection results also showed in human with different body mass index (BMI) using the biosensor. Therefore, our study demonstrated that the oriented antibody immobilization based electrochemical biosensor is an effective platform for detection of leptin in actual samples and may have broad applications in assessment of obesity diseases.