Wearable biosensors have the potential for developing individualized health evaluation and detection systems owing to their ability to provide continuous real-time physiological data. Among various wearable biosensors, localized surface plasmon resonance (LSPR)-based wearable sensors can be versatile in various practical applications owing to their sensitive interactions with specific analytes. Understanding and analyzing endocrine responses to stress is particularly crucial for evaluating human performance, diagnosing stress-related diseases, and monitoring mental health, as stress takes a serious toll on physiological health and psychological well-being. Cortisol is an essential biomarker of stress because of the close relationship between cortisol concentration in the human body and stress level. In this study, a flexible LSPR biosensor was manufactured to detect cortisol levels in the human body by depositing gold nanoparticle (AuNP) layers on a 3-aminopropyltriethoxysilane (APTES)-functionalized poly (dimethylsiloxane) (PDMS) substrate. Subsequently, an aptamer was immobilized on the surface of the LSPR substrate, enabling highly sensitive and selective cortisol capture owing to its specific cortisol recognition. The biosensor exhibited excellent detection ability in cortisol solutions of various concentrations ranging from 0.1 to 1000 nM with a detection limit of 0.1 nM. The flexible LSPR biosensor also demonstrated good stability under various mechanical deformations. Furthermore, the cortisol levels of the flexible LSPR biosensor were also measured in the human epidermis before and after exercise as well as in the morning and afternoon. Our biosensors, which combine easily manufactured flexible sensors with sensitive cortisol-detecting molecules to measure human stress levels, could be versatile candidates for human-friendly products.