Nesfatin-1 (NES1) is a potential biomarker found in serum and saliva that indicates hyperpolarization and depolarization in the hypothalamic ventricle nucleus as well as an increase in epileptic conditions. However, real-time investigations have not been carried out to detect changes in the concentration of NES1. In this study, we develop a multiscale pore contained carbon nanofiber-based field-effect transistor (FET) biosensor to detect NES1. The activated multiscale pore contained carbon nanofiber (a-MPCNF) is generated using a single-nozzle co-electrospinning method and a subsequent steam-activation process to obtain a signal transducer and template for immobilization of bioreceptors. The prepared biosensor exhibits a high sensitivity to NES1. It can detect levels as low as 0.1 fM of NES1, even in the presence of other interfering biomolecules. Furthermore, the a-MPCNF-based FET sensor has significant potential for practical applications in non-invasive real-time diagnosis, as indicated by its sensing performance in artificial saliva.