R-loop is one of the noncanonical structures of nucleic acid able to be formed during transcription. As a three-stranded nucleic acid structure, it consists of two components : one DNA:RNA hybrid, and a remnant ssDNA. Since the exposed ssDNA is frail, it could be said that there is tentative downsides in respect of genome stability. This R-loop structure, however, is not a merely malign factor which should be eliminated instantly, because it has crucial functions in some biological processes. For instance, recent studies on R-loop shed light on its positive roles like R-loop driven ATR pathway required for proper chromosome segregation. Considering its positive and negative aspects, it is important that this structure should be appropriately regulated. So, for better understanding of these regulation processes, our study has focused on observing the formation and elimination of R-loop using single-molecule FRET technique. As a result, for the first time, we pulled off observing the co-trnascriptional formation of R-loop at the single molecule level in real time. And then, we found out the detail features of this structure such as reaction to RNase H known for eliminating R-loop, relation with G-quadruplex, and formation efficiency in different sequences including trinucleotide repeat expansion sequence cuasing neurodegenerative disorders like Huntington's disease.