In this study, a highly selective Graphene oxide (GO)-Polyaniline (PANI) modified glassy carbon electrode (GCE) was fabricated and its electrocatalytic behaviour towards the electro-reduction of cortisol in human saliva was investigated. The synthesized nanocomposites were characterized using different analytical approaches. The electrochemical study of the PANI-GO modified electrode was analyzed by employing a cyclic Voltammetry (CV) study. The outcomes demonstrated a substantial enhancement in the reduction and oxidation current peaks of the modified GCE as associated to bare GCE, GO/GCE and PANI/GCE. Along with enhanced electronic properties, the biosensor displays the good selectivity and sensitivity, good reproducibility (95 %, n = 8), high stability (maintaining more than 95 % response even after 11 weeks of storage at −20C, 50 cycles), a wide linear range (100μ-10 mM) and small detection limit (30 μM). The utilization of PANI-GO in the electrode modification process may be attributed to these positive outcomes, given some of the amazing characteristics of the composite, such as its larger surface area, outstanding electrical conductivity, and good electrocatalytic behavior. Furthermore, the PANI-GO modified GCE was successfully validated by detecting cortisol in real human saliva samples using cyclic voltammetry, highlighting the effectiveness of the PANI-GO composite for non-invasive electrochemical cortisol detection.