Parathyroid hormone (PTH) is a polypeptide containing 84 amino acids secreted by cells of the parathyroid glands. Imbalances of parathyroid levels cause medical problems such as osteoporosis, mental disorders, pancreatitis, kidney stones, cancer, and other symptoms. In this study, we aimed to design an ultrasensitive electrochemical immunosensor for PTH detection. Indium tin oxide (ITO) was used as an electrode for electrochemical impedance spectroscopy (EIS) measurements. ITO sheets were modified by using 3-cyanopropyltrimethoxysilane (3-CPTMS) self-assembled monolayers (SAMs) for immobilizing the anti-PTH antibody via covalent interactions. Cyclic voltammetry (CV) and EIS methods were applied to characterize immobilization steps. Therefore, 1% was selected for an optimal concentration of silane, 10 ng/mL was selected as an optimal concentration of anti-PTH, and 30 and 45 min were selected as optimal incubation times for anti-PTH and PTH, respectively. PTH antigen was determined in the concentration range from 0.05 fg/mL to 150 fg/mL. To detect the analytical characterization of the 3-CPTMS modified immunosensor, linear range, repeatability, reproducibility, Kramers-Kronig transform, and regeneration studies were performed. Also, the shelf life of the developed biosensor was investigated. Finally, real human serum samples were analyzed with the PTH immunosensor. The results showed that the designed biosensor system has high potential for early detection for medical treatments.