The rapid and timely evaluation of the mental health of emergency rescuers can effectively improve the quality of emergency rescues. However, biosensors for mental health evaluation are now facing challenges, such as the rapid and portable detection of multiple mental biomarkers. In this study, a non-invasive, flexible, wearable electrochemical biosensor was constructed based on the self-assembly of nanomagnetic beads for the rapid detection of cortisol in interstitial fluid (ISF) to assess the mental stress of emergency rescuers. Based on a one-step reduction, gold nanoparticles (AuNPs) were functionally modified on a screen-printed electrode to improve the detection of electrochemical properties. Afterwards, nanocomposites of MXene and multi-wall carbon nanotubes were coated onto the AuNPs layer through a physical deposition to enhance the electron transfer rate. The carboxylated nanomagnetic beads immobilized with a cortisol antibody were treated as sensing elements for the specific recognition of the mental stress marker, cortisol. With the rapid attraction of magnets to nanomagnetic beads, the sensing element can be rapidly replaced on the electrode uniformly, which can lead to extreme improvements in detection efficiency. The detected linear response to cortisol was 0-32 ng/mL. With the integrated reverse iontophoresis technique on a flexible printed circuit board, the ISF can be extracted non-invasively for wearable cortisol detection. The stimulating current was set to be under 1 mA for the extraction, which was within the safe and acceptable range for human bodies. Therefore, based on the positive correlation between cortisol concentration and mental stress, the mental stress of emergency rescuers can be evaluated, which will provide feedback on the psychological statuses of rescuers and effectively improve rescuer safety and rescue efficiency.