Scaffolds are one of the key factors for the success of tissue engineering, in particular when dealing with anchorage-dependent cells. The concept of using scaffolds in tissue engineering lies in mimicking the physical, chemical and biological features of native extracellular matrix (ECM) in order to support cell function, which in turn regulates cellular microenvironment that directs cell growth and subsequent tissue formation. Nanofibers fabricated from both synthetic and natural polymers are being used as scaffolds in many tissue engineering applications. At the molecular level, native ECM is made up of a gradient of fibrous proteins and polysaccharides that are nanoscale structures. The gradient cues of ECM, directs critical cell behaviors such as alignment, motility and differentiation, particularly in the region between soft and hard tissues called interfacial tissue. Therefore, it is essential to develop gradient nanofiber scaffolds particularly for interfacial tissue engineering applications. Keeping these points in view, in this article, we review the recent developments of gradient nanofiber scaffolds, their design strategies, and their applications in tissue engineering. Copyright © 2013 American Scientific Publishers All rights reserved.