Biological processes are accomplished by bio-molecular interactions. In particular, protein-DNA interactions are fundamental and essential for numerous biological phenomena. The study of protein-DNA interactions is very important for understanding the molecular mechanisms underlying diverse cellular activities. One of best ways to examine the interplay between proteins and DNA is directly viewing the biomolecules in real time. Such a direct observation seems not to be feasible when using traditional approaches in which numerous bio-molecules are simultaneously examined in bulk; hence, only the averaged propensity of the molecules is measured. However, single-molecule techniques, which overcome the ensemble-average effect in bulk assays, allow us to detect individual biomolecules and their reactions. Among the many single-molecule methods developed until now, the DNA curtain assay provides the most straightforward platform for the protein-DNA interaction. Here, the principles of operating the DNA curtain, as well as some biological issues to which this novel assay can be applied are introduced. The DNA curtain allows us to examine how proteins function on nucleic acid substrates by integrating lipid fluidity, microfluidics, nanofabrication, and fluorescence microscopy. Using the technique, we can examine 1) specific bindings of proteins, 2) protein movements such as diffusion or active translocation, and 3) collisions between proteins on double-stranded DNA substrates. Furthermore, the single-stranded DNA curtain facilitates studies on biological processes that are mediated by single-stranded DNA.