This study examines the use of a conductive carbon fiber to construct a flexible biosensing platform for monitoring biomarkers in sweat. Cortisol was chosen as a model analyte. Functionalization of the conductive carbon yarn (CCY) with ellipsoidal Fe2O3 has been performed to immobilize the antibodies specific to cortisol. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) chemistry has been used to immobilize the antibodies onto the Fe2O3 modified CCY. Crystallinity, structure, morphology, flexibility, surface area, and elemental analysis were studied using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (FE-SEM/EDS) and Brunauer–Emmett–Teller (BET) analysis. Mechanical properties of the fiber such as tensile strength, young’s modulus have also been investigated. Under optimal parameters, the fabric sensor exhibited a good linearity (r2 = 0.998) for wide a linear range from 1 fg to 1 μg with a detection limit of 0.005 fg/mL for the sensitive detection of cortisol. Repeatability, reliability, reproducibility, and anti-interference properties of the current sensor have been investigated. Detection of cortisol levels in human sweat samples has also been investigated and the results were validated with commercial chemiluminescence immunoassay (CLIA) method.