In the neuroendocrine system, corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) play important roles in the regulation of the hypothalamic-pituitary-adrenal (HPA) system. Disorders of the HPA system lead to physiological problems, such as Addison's disease and Cushing's syndrome. Therefore, detection of CRH and ACTH is essential for diagnosing disorders related to the HPA system. Herein, receptors of the HPA axis were used to construct a bioelectronic sensor system for the detection of CRH and ACTH. The CRH receptor, corticotropin-releasing hormone receptor 1 (CRHR1), and the ACTH receptor, melanocortin 2 receptor (MC2R), were produced using an Escherichia coli expression system, and were reconstituted using nanodisc (ND) technology. The receptor-embedded NDs were immobilized on a floating electrode of a carbon nanotube field-effect transistor (CNT-FET). The constructed sensors sensitively detected CRH and ACTH to a concentration of 1 fM with high selectivity in real time. Furthermore, the reliable detection of CRH and ACTH in human plasma by the developed sensors demonstrated their potential in clinical and practical applications. These results indicate that CRHR1 and MC2R-based bioelectronic sensors can be applied for rapid and efficient detection of CRH and ACTH.