Electrospinning was patented in 1902. This technology is attracting considerable research attention as a result of increasing demand to nanoscience and nanotechnology. Nanofiber structures (from 10 nm to 100 nm or bigger) have been obtained from synthetic and natural polymers via electrospinning techniques. The resulted electrospun nanofibers are used in various areas such as public health, biotechnology, environmental engineering, textile, defense industry, and energy deposition. Among them, analytical applications have a great potential as a sorbent for chromatographic separations and a modifier of surfaces in electrochemistry or spectroscopy. In this chapter, electrospun nanofibers based separation and detection systems will be explained in detail.