In this work, a highly selective and sensitive cortisol immunosensor based on MXene and multi-walled carbon nanotubes (MXene-MWCNTs) hybrid composite was proposed. The immunosensor was developed by screen printing technique on a flexible polyethyleneterephthalate (PET) substrate followed by modification of MXene-MWCNTs and anti-cortisol antibodies with EDC/NHS coupling agent. Owing to the large surface area and excellent conductivity of MXene-MWCNTs, the immunosensor exhibited a low detection limit and a wide linear range toward cortisol from 0.1 fg/mL to 1 µg/mL, which covered the normal range of free cortisol in sweat. Additionally, the immunosensor was applied to evaluate the cortisol level in human sweat samples, and the results were validated by a commercial chemiluminescence immunoassay (CLIA) assay. The present platform provides an effective method for non-invasive monitoring of human sweat cortisol levels, which may be well-suited for hormone screenings in healthy monitoring and medical diagnosis.