A gold nanoparticle (AuNP) functionalized laser‐induced graphene interdigitated electrode (LIG‐IDE) is fabricated for the label‐free detection of cortisol. Self‐assembled monolayers of 3‐Mercaptopropionic acid immobilized on the AuNP functionalized LIG‐IDE are activated via carbodiimide chemistry for the binding of the anti‐cortisol antibody. The resulting immunosensor is tested for the specific detection of cortisol over the physiologic range (0.1 pm–100 nm) in synthetic human sweat. Electrochemical impedance spectroscopy is used to measure the change in impedance due to cortisol bonding to the LIG surface. The sensor exhibits a good linear (log(concentration)) fit over the entire concentration range (R2 = 0.9662) and a LOD of 0.0085 nm. The minimal effect of synethtic sweat shows its potential use in real‐world applications. A specificity test against similarly structured molecules demonstrates high affinity toward cortisol. Furthermore, the developed sensor exhibits excellent reproducibility with a 5.93%RSD. Given the simple and facile processability of immunosensors, the developed label‐free AuNP functionalized LIG‐based biosensing platform shows great promise as an effective and low‐cost tool suitable for healthcare monitoring and point‐of‐care diagnostics and, it can be applied as an immunosensor for other affinity‐based biosensors.