Many recent references show that living cells can form some membrane-less organelles by liquid–liquid phase separation (LLPS) of biomolecules, like proteins and nucleic acids. LLPS has been confirmed to link with many important biological functions in living cells, and one of the most important functions of biomolecular condensates is in the field of RNA transcription. Many studies confirm that mammalian RNA polymerase II (Pol II) molecules containing the CTD with different phosphorylation level are purposed to shuttle between initiation condensates and elongation condensates of RNA transcription. Traditional ensemble assays often experience difficulties in quantitively and directly recording the transient recruitment of Pol II CTD. Novel single-molecule approach — DNA curtains can be used to directly visualize biomolecular condensates formation and also recruitment of RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) at the target sites in solution and in real time. This method can offer the potential for new insights into the mechanism of gene transcription. Here, we highlight the detailed protocol of DNA curtains method for studying LLPS.