In this study, cortisol, which is a key stress hormone, could be detected sensitively via the colorimetric assay of a polycarbonate (PC) and glass substrate by the sandwich assay of cortisol monoclonal antibody (c-Mab) and cortisol specific binding aptamer (c-SBA). A highly sensitive change in colorimetry with a limit of detection (LOD) of cortisol of 100 fM could be attained on the optically transparent substrate using the antibody aptamer sandwich (AAS) assay by corresponding stacks of 5 nm gold nanoparticles (Au NPs). The Au NPs were conjugated by the c-SBA and the c-Mab was tethered on the PC and glass substrates. For the AAS method, a simple UV-Vis spectrophotometer was adopted to quantify the cortisol concentrations at an absorbance wavelength of 520 nm. Therefore, this study demonstrates the versatility of the AAS method to measure very low concentrations of cortisol in diagnostic applications.