The only solutions which showed a significantly greater fluorescence signal than the solvent were the solutions containing the TO-cortisol aptamer complex with the most significant fluorescence signal and the TO-cortisol aptamer complex in presence of hydrocortisone with the second highest signal. The remaining solutions containing the cortisol aptamer, TO, and TO + hydrocortisone, respectively, did not emit remarkable fluorescence signal compared to the solvent. Thus, it was specified that the fluorescence of TO was affected only by intercalation into the aptamer, and that this intercalation can be disturbed by the target of the aptamer, hydrocortisone.