Electrospunnanofibers have found myriad of applications from separation science to clinical translation. Electrospunnanofiber scaffolds have the benefits of unique properties such as high surface area to volume ratio, interfibrous pore sizes, strong penetrability, great deal of active sites for adsorption, excellent stability, better targeting, minimum toxicity, high drug-loading capacity, exceptional mechanical properties, flexibility in surface functionality, ease of encapsulation of drugs and bioactive compounds, suitability for thermos-liable drugs, enhanced cellular interactions, and protein absorption to facilitate binding sites for cell receptors. In the field of separation science, electrospunnanofiber scaffolds have extensively served as sorbent material for solid phase extraction techniques mainly due to the need to improve sorptive capacity and analyte selectivity. Given that almost all of the human tissues and organs are deposited in nanofibrous forms or structures, electrospunnanofibers/ nanocomposites are currently being investigated for potential clinical applications. It is noteworthy that the nanofiber fabrication technique and the material integrity are key components to obtaining clinically relevant nanofibers. Owing to the significance of fiber arrangement to nanofiber performance, electrospinning has a leading edge over other nanofiber fabrication techniques due to the ease of controlling fiber orientation, despite the inherent advantages of other conventional nanofiber fabrication techniques. The current review highlights the superb qualities of electrospunnanofibers, their various methods of fabrication, and their various applications especially in separation science and clinically. We further provided an overview of the electrospinning principles, types of electrospinning, parameters that affect the nanofibers fabrication via electrospinning, challenges, and the future directions. The advent of robotics-assisted electrospinning technique offers new opportunities for the traditional biofabrication in higher accuracy and controllability and hence will certainly drive nanotechnology from laboratory/industry toward patient care in the near future.