We used TO as a fluorescent indicator for interaction between a cortisol aptamer and its target, hydrocortisone, with the purpose of developing a diagnostic platform to determine the level of cortisol in saliva. Our in silico docking approach showed that the hydrocortisone binds in the similar pocket as found in previous literature. The mechanism was evaluated by observing a decrease in TO fluorescence based on a FID displacement assay where TO leaves the cortisol aptamer upon binding of hydrocortisone. TO showed a strong fluorescence emission upon intercalation in the cortisol aptamer. A decrease in fluorescence was observed upon addition of hydrocortisone (in HS Tris buffer) to a lowest observable concentration equal to 50 nM hydrocortisone which is relatively close to the upper boundary of cortisol in saliva. This shows the potential of using aptamers for detection of low concentrations of hormones although improvements on the sensitivity are still needed. However, it can be concluded that the TO-aptamer mechanism is not able to detect the low concentration of cortisol in saliva without prior treatment of the saliva sample to reduce the concentration of proteins in the sample as mucins interfere with the signal of TO fluorescence upon displacement.