The nonradiative relaxation mechanism of the newly synthesized hybridized-sensitive DNA probe has not been fully understood until now. In this study, the transient dark state of the probe, which is a double fluorescent dye attached to a specific DNA sequence, was investigated using a fluorescence correlation spectroscopy (FCS). The transient dark state was measured in various solvents that are known to affect the intersystem crossing or photoisomerization of the DNA probe. On the basis of the experimental results, a simplified two energy state model of the probe was constructed, and this model provides an insight into the nonradiative relaxation mechanism of the fluorophore and the applications for DNA and RNA detection. The transient dark state that was measured in a time scale of a few microseconds is a triplet state and is related to photoisomerization, viscosity, oxygen concentration, and hybridization, all of which are important parameters for cellular microscopy. The transient dark state in a time scale of a sub-microseconds is sensitively changed after the addition of target DNA. The characterization can improve the probe's capability to identify target DNA/RNA by using FCS since the triplet state that occurred after hybridization is distinctive in the time scale with that occurred before hybridization.