The aim of this work is to develop a reusable polypropylene glycol (PPG):β-cyclodextrin (βCD) biosensor for cortisol detection. To achieve the most stable support for βCD, we developed two PPG surfaces. The first surface is based on a gold surface modified with SAM of 3-mercaptopropionic acid (3MPA), and the second surface is based on a glassy carbon surface grafted with 4-carboxyphenyl diazonium salt. We characterized both surfaces by EIS, XPS, and ATR-FTIR and evaluated the stability and reusability of each surface. We found the GC-carboxyphenyl-PPG:βCD is stable for at least 1 month. We have also demonstrated the reusability of the surface up to 10 times. In detecting cortisol, we used a nonfaradaic electrochemical impedance capacitive model to interpret the surface confirmation changes. We achieved sensitive detection of cortisol in PBS buffer, urine, and saliva with limit of detection of 2.13, 1.29, and 1.33 nM, respectively.