Recent development of nanofluidic devices using nanopillars, nanowires, and nanoballs for high-performance biomolecules analysis are reviewed in this chapter. Two approaches, “top-down” fabrication techniques and “bottom-up” self-assemble techniques, were applied to construct nanospace inside microchannels, and various biomolecules including DNA, RNA and proteins were successfully separated within a few seconds. These separation techniques enabled high throughput analysis that had never achieved by natural or synthetic polymers and explored a new bioanalytical field based on molecular dynamics in nanospace. Hybrid use of the both approaches might be promising for future home diagnostic devices and clinical applications.