Dithiobis(succinimidyl propionate) (DTSP) self-assembled monolayer (SAM) functionalized gold microelectrode array has been used for covalent immobilization of monoclonal cortisol antibody (C-Mab). Binding interactions of cortisol to EA/CMab/DTSP/Au electrode was tested using impedimetric technique. This Bio-electrode was used to estimate the concentration of cortisol in samples of saliva and Interstitial Fluid (ISF). Sensors were exposed to solutions with different cortisol concentration and label-free electrochemical impedance (EIS) technique was used to make cortisol measurements. EIS results (change in charge transfer resistance, Rct) confirmed that EA/C-Mab/DTSP/Au based biosensor can accurately detect cortisol in the range of 1 pM to 1 μM has exhibit detection limit of 1 pM. In-vitro measurement of cortisol levels in saliva and ISF shows good correlation with the values obtained with commercial ELISA experiments.