Conductive nanofibers can exhibit excellent mechanical properties such as flexibility, elasticity, porosity, large surface area-to-volume ratio, etc making them suitable for a wide range of applications including biosensor development. Their large surface area provides more active sites for immobilization of large amount of bioreceptors enabling more interaction sites with the target analytes, enhancing sensitivity and detection capabilities. However, engineering conductive nanofibers with such excellent properties is challenging limiting their effective deployment for intended applications. In this research, we propose a novel approach for easy fabrication of highly conductive and flexible nanofiber leveraging the electrospinning, electroless deposition and have applied it to cortisol monitoring; a common biomarker for stress which is often quantified through enzyme-linked immunoassays using blood or saliva samples. By adopting the nanofiber sheet as a transducer for aptamer immobilization and cortisol sensing our developed biosensor was able to detect cortisol in buffer, artificial saliva, and artificial sweat within five minutes, from 10 pg/mL to 10 µg/mL (27.59 pM to 27.59 µM) with a low detection limit of 1 pg/ml (2.76 pM). The Au-coated PLA nanofiber-based electrochemical biosensor's flexibility allows for compact manufacturing, rendering it an optimal choice for integration into point-of-care testing and wearable systems.